Genotype characteristics and immunological indicator evaluation of 311 hemophagocytic lymphohistiocytosis cases in China

Abstract

Background: Primary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification.

Methods: A retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined.

Results: Among the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients (n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend (P_trend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values (P_trend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33-93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83-26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%).

Conclusions: Various immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.

Keywords: Genetic testing; Primary hemophagocytic lymphohistiocytosis; Rapid immunological indicators.

Fig. 1

Pie charts showing the proportions of patients with negative/positive genetic findings (left) and the different types of gene mutations among those with positive genetic findings (right). Hom: homozygous; hemi: hemizygous; and het: heterozygous

Fig. 2

Pie charts showing the frequencies of the gene mutations. a Frequencies of the different gene mutations associated with HLH. b Frequencies of the pathogenic genes in pHLH patients under 18 (children and adolescents) (left) and adult patients (right)

Fig. 3

Comparison of the etiology of HLH at different onset ages

Fig. 4

Gene abnormalities in different underlying diseases associated with secondary HLH

Fig. 5

Comparison of NK cell activities in patients with different types of mutations. Error bars in the scatter plot indicate the median for different groups. A Jonckheere-Terpstra test was used for statistical analysis. *P < 0.05, ** P < 0.01

Fig. 6

Comparison of CD107a in patients with different types of mutations in degranulation-related gene

Fig. 7

Determination of diagnostic efficacies of CD107a and NK cell activity assays for the presence of genetic abnormalities in patients with HLH. a Comparison of CD107a and NK cell activity assays with genetic testing results. b Empirical ROC curves for the detection of NK cell activity

Fig. 8

Diagnostic accuracy of perforin/SAP/XIAP expression for the presence of genetic abnormalities in patients with HLH. Determination of sensitivity, specificity, PPV, and NPV for low or absent (a) perforin expression to distinguish FHL-2 from all other PRF1 sequencing results; (b) perforin expression to distinguish FHL-2 from normal PRF1 sequencing results; (c) SAP expression to distinguish patients with abnormal SH2D1A sequencing results from normal ones; and (d) XIAP expression to distinguish patients with abnormal XIAP/BIRC4 sequencing results from normal ones. PPV, positive predictive value; NPV, negative predictive value