Profiles of Cytotoxic Molecules In Mature NK Cell Neoplasms

Proliferative disorders of mature natural killer (NK) cells are classified based on their pathologic and clinical characteristics and divided into three types in WHO 2008 classification, extranodal NK cell lymphoma, nasal type (ENKL), aggressive NK cell leukemia (ANKL) and chronic NK lymphocytosis (CNKL). ENKL and ANKL show strong association with EBV and are clinically aggressive, but CNKL usually dose not and takes insidious course.

Cytotoxic molecules are biological important factors possibly associated with tissue injuries in NK cell neoplasms. There are several cytotoxic molecules residing in the cytotoxic granules, including granulysin; a member of saposin-like lipid binding proteins, granzyme B and perforin. However, their relevance in NK cell neoplasms is still not clear.

We investigated the relationships of profiles of cytotoxic molecules with NK cell neoplasms.

Patients with ENKL, ANKL and CNKL were studied. The diagnoses were made according to the WHO classification. Healthy controls and the patients with T-cell large granular lymphocyte leukemia were also included for serum granulysin evaluation.

Concentration of serum granulysin was measured with ELISA. Histologic examinations of granulysin, granzyme B, perforin and TIA-1 in paraffin-embedded tissues were made by immunostaining with antibodies corresponding to the antigens. EBV viral load in the whole blood and tissues were detected by real time PCR and EBER in situ hybridization, respectively.

Total 38 patients (pts) with NK cell neoplasms, including 17 ENKL, 9 ANKL, and 12 CNKL, were recruited. Median age of the patients was 42 years, 36 years, and 72 years respectively. In ENKL patients, 16 pts were nasal lymphoma and 5 pts were at stage IV. Most of the patients with ANKL showed hepatosplenomegaly, although a few CNKL pts with hepatomegaly were recognized. Liver dysfunction were observed in 29% of ENKL, 88% of ANKL, and 33% of CNKL pts. EBV was positive for all ENKL and ANKL pts, and negative for pts with CNKL.

Mean serum granulylin levels were 2.52 ng/ml, 64.2 ng/ml, and 14.43 ng/ml, in ENKL, ANKL, and CNKL, respectively, (Fig.1) which were significantly higher than controls (p<0.01). ANKL pts showed the highest serum granulysin levels compared to ENKL and CNKL, and higher than those of stage IV ENKL pts (p=0.04). Serum granulysin levels are also correlated with the EBV viral load in the whole blood of EBV-positive ENKL and ANKL pts (p=0.02). After achievement of complete response, elevated serum granulysin returned to within the range of controls. Pts with higher serum granulysin tended to show liver dysfunction.

Immunohistochemical studies of the cytotoxic molecules showed that all ENKL pts were positive for granzyme B and perforin. In contrast, granzyme B and perforin are positive in 57% and 85%, respectively in ANKL pts. 60% of CNKL pts were positive for granzyme B and perforin. Granulysin was positive in 50%, 29% and 25% in ENKL, ANKL and CNKL, respectively. TIA-1 was positive in all pts. From these results, three types of histologic cytotoxic molecule patterns were classified, which are granzyme B+, perforin+, granulysin+ type (n=10), granzyme B+, perforin+, granulysin-negative type (n=10), and granzyme B weakly+, perforin weakly+, granulysin-negative type (n=5). Pts in which proliferating NK cells were histologically negative for granulysin showed lower or normal serum granulysin values.

Considerable heterogeneities were recognized in NK cell neoplasms in respect to cytotoxic molecules that are associated with certain clinical characteristics of NK cell proliferative disorders. In addition, serum granulysin might serve as a novel biomarker for NK cell lymphoma/leukemia.

No relevant conflicts of interest to declare.