Comprehensive Analysis and Summary of the Value of Immunophenotypes of Mature NK Cell Tumors for Differential Diagnosis, Treatment, and Prognosis

Abstract

Background: Few studies have been performed to comprehensively analyze and summarize the immunophenotype and differential diagnosis of mature NK cell tumors, and there is often overlap between tumorigenic and reactive NK cell phenotypes. Furthermore, the impact of different phenotypes on patient prognosis has rarely been reported.

Methods: The degree of expression of extracellular and intracellular markers of NK cells in each group was compared by FCM, and the differences in expression of various markers among different disease groups and their impact on prognosis have been analyzed and summarized.

Results: Compared with normal NK cells, tumor cells of ANKL and ENKTL had characteristics of being more activated and progressive with larger FSC, in contrast to NK-CLPD and RNKL. Differential diagnoses with RNKL, ANKL, and ENKTL have broader FCM clues. In contrast, the phenotypes of NK-CLPD and RNKL are not significantly different, and consistent phenotypic abnormalities require ongoing monitoring to confirm malignant clones. The sensitivity of differentiating malignant NK cells from reactive NK cells by KIRs alone was poor. The clustering results showed that CD5, CD16, CD56, CD57, CD94, CD45RA, CD45RO, HLA-DR, KIRs, Granzyme B, Perforin and Ki-67 were differentially distributed in the expression of three NK cell tumors and reactive NK cell hyperplasia, so a comprehensive judgment using a wide range of antibody combinations is required in disease staging diagnosis. The tumor cell loads in BM and PB were also compared, and there was a clear correlation between the two. Moreover, the sensitivity of PB for monitoring tumor cells was up to 87.10%, suggesting that PB could be used as an alternative to BM for the diagnosis and screening of NK cell tumors. Analysis of the phenotypic impact of ENKTL patients on prognosis showed that those with CD7 and CD45RO expression had a poor prognosis, while those with positive KIRs had a better prognosis.

Conclusion: This study systematically characterized the FCM of mature NK cell tumors, emphasizing the importance and clinical value of accurate immunophenotyping in diagnosing, classifying, determining prognosis, and guiding treatment of the disease.

Keywords: FCM immunophenotype; diagnosis and treatment; mature NK cell tumors; prognosis; reactive NK cell hyperplasia.

Figure 1

Expression of different extracellular and intracellular markers in ANKL, ENKTL, NK-CLPD, RNKL, and normal NK cells. (A) Cell surface pan-lineage-associated markers were analyzed by flow cytometry. Representative FACS plots show the expression of CD2, sCD3, CD4, CD5, CD7, and CD8 in tumorigenic NK cells, reactive NK cells, and normal NK cells. (B) Analysis of lineage-specific markers of NK cells. Representative FACS graphs show the expression of CD16, CD56, CD57, and CD94 in each group of NK cells. (C) Analysis of functional markers of NK cells. Graphs show the expression of Granzyme B as well as Perforin in each group of NK cells. (D) Representative FACS graph showing the expression of Ki-67, a proliferation marker for each group of NK cells. (E) Representative FACS graphs showing the expression of the depletion marker PD-1 in each group of NK cells. (F) Representative FACS graphs showing the expression of activation markers CD30, CD38, and HLA-DR in each group of NK cells. (G) Analysis of the differences between CD45RA and CD45RO in aberrant NK cells. Representative FACS graphs show the expression of abnormal indicator markers CD45RA and CD45RO in each group of NK cells. (H) The Clonality of NK cells was determined by analyzing the expression distribution of KIRs. Representative FACS graphs show the expression of CD158a, CD158b, CD158e, and CD158i in each group of NK cells. Red scatter dots represent tumorigenic NK cells, yellow scatter dots represent reactive NK cells, and green scatter dots represent normal NK cells. The blue scatters on the graph represents other normal T lymphocytes in the background and can be used as a negative or positive control for marker expression.

Figure 2

FSC and SSC characteristics of NK cells in each group. (A) FSC of tumor cells in ANKL and ENKTL were significantly higher than those of normal NK cells and reactive NK cells. There was no significant difference in SSC between the NK cells of each group. (B) Morphologically tumorigenic NK cells in ANKL and ENKTL were significantly larger with or without granules in the cytoplasm (a, ANKL; b, ENKTL; c, NK-CLPD; d, RNKL; e, NNK; 1000×) (**p<0.01, ***p<0.001). ns, no significant difference.

Figure 3

Phenotype-based cluster analysis of ANKL, ENKTL, NK-CLPD and RNKL. (A) Clustering of 20 immunomarkers of NK cells from patients with ANKL (n = 14), ENKTL (n = 23), NK-CLPD (n = 8) and RNKL (n = 10) Graph. The horizontal axis is for each individual patient and the vertical axis is for the immune markers. (B) Correlation matrix of the 20 immunomarkers. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

The differences in expression based on the specific markers between clonal NK cells and normal NK cells. (A) Abnormal expression or loss of CD2, CD4, CD5, and CD7 (The phenotype of normal NK cells is usually CD2+CD4-CD5-CD7+). (B) Homogeneous attenuation or loss of CD16 (normal NK cells CD16+). (C) Strong expression of CD56 (stronger than normal NK cell CD56 expression level). (D) Loss of CD57 homogeneity (heterogeneous scattered expression of CD57 in normal NK cells). (E) CD45RA-/CD45RO+ or CD45RA+dim/CD45RO+dim (normal NK cells express CD45RA+CD45RO-). (F) Positive or weakly positive expression of HLA-DR (normal NK cells do not express HLA-DR). (G) None KIRs expression (normal NK cells have scattered expression of all subtypes of KIRs). (H) Perforin or Granzyme B is not expressed (normal NK cells express Perforin+/Granzyme B+). (I) Ki-67 or CD279 (PD-1) or CD30 positive expression (normal NK cells do not express any of the three). Red scattered dots in the graph represent clonal NK cells and green scattered dots represent normal NK cells.

Figure 5

Correlation and homology of tumorigenic NK cell load in the bone marrow and peripheral blood. (A) Correlation analysis of the proportion of tumorigenic NK cells occupying lymphocytes in the bone marrow and peripheral blood, respectively, showed a positive correlation (p < 0.0001). (B) Correlation analysis of the proportion of tumor NK cells occupying nucleated cells in the bone marrow and peripheral blood, respectively, showed a positive correlation (p < 0.0001). (C) Morphological analysis verified that tumor cells in the bone marrow and peripheral blood had similar morphology (a, BM; b, PB; 1000×).

Figure 6

Prediction of survival of ENKTL by immunophenotype (n = 23). (A) Effect of CD7 expression on survival time of patients. (B) Effect of CD45RO expression on survival time of patients. (C) Effect of expression of KIRs on survival time of patients.