Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes

Abstract

Background: The aim of this study was to investigate the prognostic effect of tumour-infiltrating lymphocytes (TILs) in serous stage III ovarian carcinoma to determine TIL clonality and to correlate this to Her2/neu expression.

Methods: Formalin-fixed and paraffin-embedded ovarian carcinomas were examined for CD20-, CD3-, CD4- and CD8-positive lymphocytes (n=100), and for Her2/neu-positive tumour cells (n=55/100) by immunohistochemistry. Clonality analysis was carried out by T-cell receptor gamma (TCRgamma) gene rearrangements (n=93/100). Statistical analyses included experimental and clinico-pathological variables, as well as disease-free (DFS) and overall (OS) survival.

Results: CD20-positive B lymphocytes were present in 57.7% (stromal)/33.0% (intraepithelial) and CD3-positive T lymphocytes in 99.0% (stromal)/90.2% (intraepithelial) of ovarian carcinomas. Intraepithelial CD3-positive T lymphocytes were correlated with improved DFS in optimally debulked patients (P=0.0402). Intraepithelial CD8-positive T lymphocytes were correlated with improved OS in all optimally debulked patients (P=0.0201) and in those undergoing paclitaxel/carboplatin therapy (P=0.0092). Finally, rarified and clonal TCRgamma gene rearrangements were detected in 37 out of 93 (39.8%) and 15 out of 93 (16.1%) cases, respectively. This was marginally associated with improved DFS (P=0.0873). Despite a significant correlation of HER2/neu status and intraepithelial CD8-positive lymphocytes (P=0.0264), this was non-directional (R=-0.257; P=0.0626).

Conclusion: Improved survival of ovarian cancer patients is related to the infiltration, clonal selection and intraepithelial persistence of T lymphocytes.

Figure 1

Summary of lymphocyte infiltration in ovarian carcinomas. The graphs provide the distribution of immunohistochemical scores, that is, lymphocyte infiltration (given as the percentage of cases with each IHC score on the y axis; Materials and Methods) in non-neoplastic ovaries (A), as well as for stromal (B) and intraepithelial lymphocytes infiltration (C) in ovarian carcinomas. Note the increased presence of CD3-positive as compared with CD4- and CD8-positive T lymphocytes in ovarian carcinomas (shift of bars to right; refer to text for P-values).

Figure 2

Representative immunohistochemical staining of lymphocytes in ovarian carcinomas. (A) Provides representative serial sections of an ovarian carcinoma stained for CD20, CD3, CD4 and CD8, with IHC scores given below the photographs (magnification × 100). (B) Shows representative IHC stainings of CD8-positive T lymphocytes in six cases, with high (case #1, 2, 3 (left)) and low (case #4, 5, 6 (right)) intraepithelial T-lymphocyte infiltration (magnification × 100), with the IHC score given beside the photographs. Note that two cases exhibited rarified/clonal (case #1) and polyclonal (case #4) T-cell receptor γ (TCRγ) rearrangements (refer to Figure 4A).

Figure 3

: Correlation of CD3- and CD8-positive T-lymphocyte infiltration of stage III ovarian carcinomas with clinical follow-up. The panels provide results of Kaplan–Meier analyses for overall survival with respect to intraepithelial (A and B) and stromal (C and D) CD8-positive T lymphocytes in stage III patients with optimal debulking (A and C), as well as for stage III patients with optimal debulking and adjuvant paclitaxel/carboplatin chemotherapy (B and D). Significant correlation to improved overall survival was only seen for increasing numbers of intraepithelial CD8-positive T lymphocytes (A and B; refer also to main text). Coding: red lines=IHC score 0 (no tumour-infiltrating lymphocytes (TILs)), blue lines=IHC score 1 (<5 TILs) and green lines=IHC scores 2+3 (>5 TILs).

Figure 4

Analysis of T-cell receptor γ (TCRγ) gene rearrangements and association with survival. (A) Provides electropherogram traces of the PCR-based TCRγ gene rearrangement analysis (Materials and Methods) (van Dongen et al, 2003) for Vγ1-8,10 (left) and Vγ9,11 (right) in four ovarian carcinomas (cases 1, 4, 7 and 8; cases 1 and 4 are the same as in Figure 2B) and two non-neoplastic ovaries (cases 9 and 10). Cases 1 and 7 show polyclonal TCRγ gene rearrangements for Vγ1-8,10 (left) and rarified/clonal TCRVγ gene rearrangements for Vγ9,11 (right, black arrows). Cases 8 and 4 show polyclonal TCRγ gene rearrangements for Vγ1-8,10 (left) and oligoclonal/rarified TCRVγ gene rearrangements for Vγ9,11 (right, black arrowheads). The non-neoplastic ovaries (cases 9 and 10) show polyclonal/oligoclonal TCRγ gene rearrangements. The score for intraepithelial CD3- and CD8-positive T-cell infiltration of each case is given beside the corresponding graphs. In the eletropherogram traces, the top x axis indicates the size range of PCR products in base pairs and the y axis indicates the intensity of the PCR product. Blue lines/peaks represent labelled, TCRγ-specific PCR products and red lines/peaks represent the standard size marker. (B) Provides the results of Kaplan–Meier analyses for disease-free (left) and overall (right) survival of all cases with polyclonal (red line) or rarified/clonal (blue line) TCRγ gene rearrangements. Note the trend towards early improved disease-free survival for cases with rarified/clonal TCRγ gene rearrangements.