Clinicopathological significance of CD28 overexpression in adult T‐cell leukemia/lymphoma

Abstract CD28, one of the costimulatory molecules, has a pivotal role in T‐cell activation, and its expression is strictly regulated in normal T cells. Gain‐of‐function genetic alterations involving CD28 have been frequently observed in adult T‐cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non‐overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86‐positive non‐neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non‐overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.

this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with nonoverexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86-positive non-neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non-overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had

| INTRODUC TI ON
CD28 is a costimulatory molecule expressed on T cells which has a pivotal role in T-cell biology. In normal T cells, CD28 is expressed on the cell surface, providing an essential costimulatory signal for T-cell activation upon ligation by CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APCs). After ligation, the accessory molecule cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is upregulated. Normally, CTLA4 transmits negative signals to T cells.
Because CTLA4 binds to CD80 and CD86 with significantly higher affinity than to CD28, when CTLA4 is upregulated, CD28 is immediately downregulated through a mechanism of competitive inhibition of the ligands to CD28 by CTLA4. [1][2][3][4] The importance and influence of CD28 signaling in T-cell activation was dramatically demonstrated in a clinical trial. After receiving injections of the humanized monoclonal CD28 agonist antibody TGN1412, the six volunteers became desperately ill, had multiple-organ failure, and were transferred to an intensive care unit with what has been described as a form of cytokine release syndrome. 5,6 In this context, T-cell-activating alterations to the CD28 gene have been reported in some types of peripheral T-cell malignancies such as adult T-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma (PTCL), not otherwise specified, and cutaneous T-cell lymphoma. [7][8][9][10][11][12][13][14] ATLL is a peripheral T-cell neoplasm caused by human T-cell lymphotropic virus type-1 (HTLV-1) and has a poor prognosis. [15][16][17][18] We previously reported that CD28 gene-related alterations were more frequent in this tumor, compared with other types of peripheral T-cell neoplasms, and that these alterations were associated with a worse prognosis in ATLL patients. 19 Genetic alterations involving CD28 include gene fusions such as CTLA4-CD28 and inducible T-cell costimulator (ICOS)-CD28, activating single-nucleotide variants (SNVs) such as F51I/V, D124V/E, or T195I/L/P, and copy number variations (CNVs) such as gain and amplification. Of these abnormalities, CD28 gene fusions and CNVs may not only confer continuous, prolonged, and enhanced CD28 signaling to the downstream pathways [7][8][9][10][11]20 but also are most likely to induce overexpression of the CD28 molecule. In fact, in other PTCL tumors, it has been reported that gene fusions related to CD28 were associated with CD28 overexpression, 8,21 probably because the expression of CD28 transcripts may be driven by ICOS or CTLA4 promoters.
In addition, there have been recent reports of ectopic expression of CD80 and CD86 on tumor cells in some ATLL cases, although these molecules were originally expressed on APCs and not on T cells. 22,23 These observations suggest that intracellular and intercellular interactions via the CD80/CD86-CD28 pathway may exist and contribute to ATLL pathogenesis.
The aims of this study were to clarify the clinicopathological and prognostic significance of overexpression of CD28, CD80, and CD86 in ATLL and to examine the association between the expression of these molecules and the genetic status of CD28.

| Immunohistochemistry for CD28, CD80, and CD86
Immunohistochemistry for CD28 was performed on formalinfixed paraffin-embedded (FFPE) sections of the affected tissues of ATLL patients, using a BOND-III fully automated IHC and ISH staining system (Leica Biosystems) and a BOND polymer refine detection kit (Leica Biosystems). The rabbit anti-human CD28 monoclonal antibody (clone, EPR22076; Abcam) used was diluted to 1:50 in BOND primary antibody diluent (Leica Biosystems), no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.

K E Y W O R D S
adult T-cell leukemia/lymphoma, CD28, CD80 (B7-1), CD86 (B7-2), costimulatory molecule, genetic alterations, immunohistochemistry, overexpression, prognosis and BOND epitope retrieval solution 2 (Leica Biosystems) was employed for heat-induced antigen retrieval. Areas showing representative immunohistochemical CD28 expression on the cytomembrane in each section were chosen, and the expression was evaluated semiquantitatively by the signal intensity on the tumor cells. Using a scoring scale of 0, 1+, 2+, and 3+, as described in a previous study, 33 we conducted CD28 immunostaining scoring as follows: 0, no staining; 1+, complete or incomplete membrane staining that is weak or only faintly perceptible; 2+, complete membrane staining of moderate intensity; and 3+, complete membrane staining of strong intensity. Scoring of CD28 expression was performed by experienced hematopathologists (A.M. and H.I.), and when the scoring was discordant, a consensus score was reached. The cutoff point was set between 0/1+ and 2+/3+ for CD28 expression, as this cutoff point allowed for the best segregation into prognostic groups. Based on these observations, we defined ATLL cases that were scored as 0 or 1+ as lower CD28 expressers and those that were scored as 2+ or 3+ as CD28 overexpressers.
We also performed immunohistochemistry for CD80 and CD86 and evaluated their expression on tumor cells in the same manner.
In addition, non-neoplastic cells expressing CD80 or CD86 (CD80/ CD86) in the tumor microenvironment were counted in three representative high-powered fields, and the average number for each sample was calculated, respectively. Microenvironmental expression was defined as positive if 10 or more non-neoplastic cells expressing CD80/CD86 were observed per high-powered field. 23 CD80/CD86 expression on microenvironmental cells was distinguished from that on tumor cells by cytomorphology using such parameters as a low nuclear/cytoplasmic ratio and the presence of nuclei without atypia. All types of microenvironmental cells, including macrophages, dendritic cells, and others, were counted.

| RNA extraction and quantitative RT-PCR for CD28
A real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for CD28 mRNA was carried out with a QuantStudio™ 12K Flex real-time PCR system (Thermo Fisher Scientific) using GeneAce SYBR ® qPCR Mix α (NIPPON GENE Co., Ltd.) according to the manufacturer's instructions. Details are available in the supporting information file. 34,35

| Detection of CD28 gene-related activating alterations
Details are available in the supporting information file. 19

| Detection of CCR4 protein expression and CCR4 gene mutations
Details are available in the supporting information file. 33,36

| Statistical analysis
Details are available in the supporting information file.

| Clinical and genetic features of ATLL patients
The cohort of the present study consisted of 120 ATLL patients, whose clinical characteristics are summarized in Table S1. There Collectively, alterations of a type involving the CD28 gene were found in 37 cases (31%) (data not shown).

| CD28 mRNA levels in ATLL cases
To assess CD28 mRNA levels, real-time quantitative RT-PCR analysis was performed in quantifiable cases (n = 112). As shown in Figure 3A, relative CD28 mRNA levels were significantly higher in CD28 protein overexpressers than in non-overexpressers (median, 149 vs 40, respectively, P < .0001). Furthermore, relative CD28 mRNA levels were positively associated with the degree of the CD28 protein expression score ( Figure S3). Relative CD28 mRNA levels were significantly higher in ATLL cases with CD28 generelated alterations than in those without (median, 144 vs 47, respectively, P = .007; Figure 3B).

| Survival of ATLL patients who received mogamulizumab-containing treatment but not allogeneic HSCT, stratified by CD28 or CD80/CD86 protein expression
We next investigated the survival of patients who received mogamulizumab-containing regimens but not allogeneic HSCT. data not shown). The 5-year survival from the day of antibody administration to the three patients with an indolent variant could not be estimated. All three patients were assigned to the CD28 overexpresser group, and two died within 3.5 years after the initiation of mogamulizumab treatment. As a close association between CCR4 gene mutations and a superior responsiveness of mogamulizumab was reported in ATLL patients, 36 we also performed survival analyses for the patients receiving mogamulizumab independent of their CCR4 gene status. As a result, we found that among patients with CCR4 mutations who received mogamulizumab-containing regimens but not allogeneic HSCT (n = 12), the 5-year survival from the day of the first dose of antibody for CD28 overexpressers and nonoverexpressers was 28.6% (95% CI, 30.9%-97.3%) and 80.0% (95% CI, 24.4%-75.6%), respectively (not significantly different, P = .101; Figure 5G). Among patients without CCR4 mutations who received a mogamulizumab-containing regimen but not allogeneic HSCT (n = 35), the 5-year survival from the day of the first dose of antibody for CD28 overexpressers and non-overexpressers was 0.00% (95% CI, not reached) and 45.5% (95% CI, 23.3%-69.7%), respectively (also not significantly different, P = .113; Figure 5H). As for CD80/ CD86 expression on tumor cells, the 5-year survival from the first dose of antibody in the overexpressers was 75.0% (n = 5, 95% CI, 23.8%-96.6%), again not significantly different from that (33.0%) of the non-overexpressers (n = 39, 95% CI, 18.4%-51.8%, P = .186; Figure S4D).   (Table 3).  Table S3, and HR, 1.964, 95% CI, 1.014-3.801; Table   S4, respectively).

| D ISCUSS I ON
In the current study, we examined CD28 and CD80/CD86 expression in 120 ATLL cases and correlated the expression with the clinical and genetic features and prognosis of these patients. We demonstrated that CD28 overexpression is an independent prognostic factor for a worse prognosis in ATLL patients.
The importance of aberrant CD28 overexpression has been well recognized in multiple myeloma (MM). In this tumor, CD28 overexpression is one of the major prognostic predictors for a poor clinical outcome following high-dose chemotherapy. [37][38][39] Furthermore,