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in some tumors, which plays an important role in immune escape and survival of tumors.11 Kataoka et al. found that more than half of ATLL patients had alterations in HLA class I genes.12 We have previously reported that ATLL expressing HLA class I/β2M had a good clinical outcome.13
Programmed cell death-1 (PD-1) blockade therapy is effective in various recurrent/refractory tumors. PD-1 is expressed on activated T cells and binds to PD-L1 on tumor cells. Binding of PD-L1 to PD-1 suppresses production of cytokines and inactivates T cells.14,15 HLA class I plays a key role in PD-1 blockade therapy because activated T cells attack tumor cells by recognizing neoantigens presented by HLA class I on tumor membranes. In fact, an interaction between neoantigens and neoantigen-specific T cells via HLA class I has been reported in patients with malignant melanoma who received PD-1 blockade therapy.16
It has been hypothesized that tumors lacking HLA class I and/or β2M could be resistant to PD-1 blockade thera- py.17 However, a favorable clinical response to PD-1 block- ade has been observed in patients with Hodgkin lym- phoma, who usually lack HLA class I expression due to the loss-of-function mutation of β2M.18,19 From the stand- point of immunotherapy, these results are indicative that other immunological mechanisms exist, at least among patients with Hodgkin lymphoma.
HLA class II is expressed on antigen-presenting cells including B cells, dendritic cells, and macrophages. HLA class II presents antigens to CD4+ T cells.
HLA class II expression has been reported to be associ- ated with a favorable prognosis in squamous cell carcino- ma of the larynx, colorectal cancer, and diffuse large B-cell lymphoma.20-22 In contrast, HLA class II expression was not significantly associated with prognosis in malignant melanoma, Hodgkin lymphoma, and lung cancer treated with standard chemotherapies.23-25
Roemer et al. recently reported that HLA class II expres- sion is more important than HLA class I expression for PD-1 blockade therapy in recurrent/refractory Hodgkin lymphoma.26 Although the precise mechanism remains unknown, it has been suggested that an interaction between HLA class II-expressing lymphoma cells and CD4+ T cells could contribute to tumor immunity in Hodgkin lymphoma.
The prognosis of ATLL patients treated with standard chemotherapy remains poor. PD-1 blockade could be a good therapeutic strategy; however, a clinical trial showed that PD-1 blockade in ATLL patients induced disease pro- gression.27 For a better understanding of tumor immunity associated with PD-L1/PD-1 in ATLL, we examined the clinico-pathological effect of HLA class II expression in ATLL and the correlation of HLA class II to HLA class I/β2M and PD-L1/PD-1 expression.
Methods
Patients and samples
All patients included in this study were the same as those in our previous reports.10,13 Fifty-nine patients were derived from the International Peripheral T-Cell Lymphoma Project, and 73 were newly diagnosed with ATLL in Kurume University between 2006 and 2012.28 A tissue microarray including 132 samples in formalin- fixed paraffin-embedded blocks was created. The tissue microar- ray specimens used in this study were the same as those examined in our previous studies.10,13 The use of patients’ specimens and clin-
ical data was approved by the Research Ethics Committee of Kurume University and was carried out in accordance with the Declaration of Helsinki.
Immunohistochemistry
Immunohistochemistry was performed as previously described.10,13 The following antibodies were used: HLA class II (CR3/43 M0775, DAKO, Glostrup, Denmark), HLA class I (EMR8- 5; Abcam, Tokyo, Japan), β2M (HPA006361; Sigma-Aldrich, Tokyo, Japan), PD-L1 (EPR1 161(2); Abcam), and PD-1 (NAT105, Abcam).
As in our previous studies, PD-L1 expression was determined on neoplastic cells (nPD-L1) and microenvironmental stromal cells (miPD-L1). nPD-L1 was considered positive when 50% or more neoplastic cells were positive for PD-L1.10 miPD-L1 were distin- guished from nPD-L1 by irregular shaped morphology, low nuclear/cytoplasmic ratio, and nuclei without atypia (Online Supplementary Figure S1A, S1B).10 miPD-L1 was defined as positive in patients with ten or more PD-L1+ non-neoplastic stromal cells per high power field.10 Other markers, including HLA class II, were considered positive when 30% or more tumor cells were positive. PD-1+ tumor-infiltrating lymphocytes were detected as small lym- phocytes without atypia and were distinguished from PD-1+ tumor cells (Online Supplementary Figure S1C, S1D). PD-1+ tumor- infiltrating lymphocytes were counted in five representative high- power fields, and the average number for each sample was calcu- lated as previously reported.10
Statistical analysis
haematologica | 2019; 104(8)
HLA class II is a good prognostic factor in ATLL
Clinico-pathological characteristics were compared by the χ2 test or Fisher two-sided exact test. Wilcoxon rank sum test was performed to compare age and PD-1+ tumor-infiltrating lympho- cyte counts. Overall survival was defined as the period between the day of diagnosis and the day of death or the day of last follow up. Overall survival was analyzed by the Kaplan–Meier method, and the log-rank test was performed to determine significant dif- ferences. Univariate and multivariate Cox proportional regression models were used to examine the proposed prognostic factors. All statistical analyses were performed using JMP version 11.0. P val- ues <0.05 were considered statistically significant.
Results
Clinico-pathological characteristics
The participants’ clinical information is summarized in Table 1. The median age of the participants was 67.5 years (range, 35-90), and the male to female ratio was 75:57. Seventy-eight of 126 (61.9%) patients had a high International Prognostic Index (high or high-intermediate), and 50/129 (38.8%) patients had high scores in the Japan Clinical Oncology Group Prognostic Index (JCOG-PI).29
According to the Shimoyama classification, most cases were acute type (53/108, 49.1%) or lymphoma type (52/108, 48.1%). Only three cases (2.8%) were smoldering ATLL and no cases of chronic ATLL were included in this study. Most patients received chemotherapy (115/131, 87.8%); radiation (14/129, 10.9%) and allogenic stem cell transplantation (17/129, 13.2%) were chosen in fewer cases. The chemotherapy regimens were not significantly different between the HLA class II-positive and negative groups (Online Supplementary Table S1). No PD-1 blockade therapy was given. Of the 115 patients, 32 (27.8%) achieved a complete remission. The median follow-up period was 10.96 months (range, 0.03-114.8 months).
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