Since NRP-1/CD304 plays an important role in angiogenesis,3-5 it is a promising target for anti-angiogenesis treatment strategies and many scholars have studied its application in acute leukemia.17-20,30 Kreuter et al. firstly evaluated the role of angiogenic factors in AML by detecting the expression of NRP-1/CD304 in 76 cases of AML by immunohistochemical analysis. They found that NRP-1/CD304 was overexpressed in all cases compared with normal bone marrow, and there was a significant negative correlation between survival and NRP-1/CD304 expression. In addition, the authors also found no correlation between the levels of NRP-1/CD304 expression and AML subtype or karyotype.17 Other researchers used FCM to detect the expression of NRP-1/CD304 in BPDCN, AML, B-ALL, and they found that NRP-1/CD304 was positive in varying degrees,13,22 which was similar to our study. It had been verified that NRP-1/CD304 was a very useful and dependable marker for the MRD assessment of B-ALL because it was overexpressed in B-ALL cells compared with normal precursor B cells.27-29,30 However, there are no comprehensive and detailed reports on the expression of NRP-1/CD304 in other common hematological diseases besides BPDCN, AML and B-ALL, such as T-ALL, B-NHL, T/NK-cell lymphoma and plasma cell neoplasms. Also, the application value of NRP-1/CD304 in these diseases is unknown. Therefore, it is necessary to conduct a comprehensive study to evaluate the flow application prospects of NRP-1/CD304.
According to the guidelines of the literature, pDCs in the specimens were used as the positive expression control of NRP-1/CD304, and mature lymphocytes were used as the negative expression control. Based on this, we found that the overall positive rate of NRP-1/CD304 in all specimens was 14.81% (44/297), and the positive rate of NRP-1/CD304 in descending order was as follows: BPDCN (6/6, 100%), B-ALL (48.61%, 35/72), AML (3/67, 4.48%), which were similar to those reported in other literatures.22,27,29,31 Nevertheless, NRP-1/CD304 was not positive in other hematolymphoid neoplasms (T-ALL, B-NHL, T/NK-cell lymphoma and plasma cell neoplasms). These results indicated that NRP-1/CD304 had no obvious value in flow detection and follow-up study in the above hematological diseases, and it was not necessary to include NRP-1/CD304 in the panel design of immunophenotyping and MRD detection protocols. In addition, we also found that NRP-1/CD304 was not positively expressed on normal granulocytes and monocytes in the specimens, and the intensity of expression was comparable to that of normal precursor B cells (results not shown), which was rarely reported in other literature.
The application value of NRP-1/CD304 in BPDCN, AML and B-ALL was worthy of in-depth study. Further analysis found that NRP-1/CD304 had higher sensitivity (up to 100%) but lower specificity (87.12%) in the diagnosis of BPDCN. Although NRP-1/CD304 only accounted for 13.64% of PPV diagnosed by BPDCN, NPV reached 100%. These results suggested that the positive expression of NRP-1/CD304 alone cannot determine the differentiation of plasmacytoid dendritic cells, and should be combined with other characteristic PDCs immunophenotypes (such as CD4+, CD56+, CD123high, CD303+, and lack of Lineage-specific antigens) for comprehensive diagnosis.10,13,32,33 However, negative expression of NRP-1/CD304 strongly suggested that the case could not be diagnosed as BPDCN. This conclusion was consistent with other literatures9-11,13 and our actual experience. Additionally, we found that the PPV of NRP-1/CD304 for B-ALL (79.55%) was much higher than that of BPDCN (13.64%) and AML (6.82%), and the NPV for B-ALL reached 85.60%. These results indicated that NRP-1/CD304 had a promising value in the diagnosis and therapy monitoring of B-ALL, which was agreed with other reports.27,29,31 We did not assess the utility of NRP-1/CD304 for the MRD of B-ALL as it was beyond the scope of this study. In this study, the ROC curve was used to evaluate the diagnostic efficacy of NRP-1/CD304 positive diseases including BPDCN, B-ALL and AML. The results showed that the highest diagnostic efficacy was BPDCN (AUC: 0.936), followed by B-ALL (AUC: 0.723), and NRP-1/CD304 had no significant diagnostic value for AML (AUC: 0.435). We noted an imbalance in the number of cases between the BPDCN and B-ALL groups due to the rare incidence of BPDCN. Small sample size of BPDCN would lead to the deviation of sensitivity and specificity. The factors affecting the predictive value (PV) are the prevalence, sensitivity and specificity of the disease. Therefore, the deviation of sensitivity and specificity caused by the small size of BPDCN group would directly lead to the bias of PV. In addition, with the increase of prevalence, the PPV would show an upward trend, while the NPV would show a downward trend under the condition of unchanged sensitivity and specificity. Therefore, higher incidence of B-ALL and lower incidence of BPDCN also affected their respective PPVs. Expanding the sample size of BPDCN can make the statistical results more reliable, whereas the influence of the relatively stable prevalence difference between BPDCN and B-ALL disease itself on PV cannot be eliminated.
We also observed that male-to-female ratio was 1:5 as well as the median age was 34 years (range 12-70) in the BPDCN group of our study, which was inconsistent with literature reports. Many studies had shown that BPDCN was a rare, male-predominant hematologic malignancy among older patients.8,9,14,15,34 The male-to-female ratio was approximately 3.3:1 and the median age at diagnosis was 61-67 years without racial or ethnic predilection.35 Although a study based on a comprehensive literature database of cases identified a more equal male to female prevalence among patients younger than 40 years,36 there was a cohort bias leaded to the female-predominance and relatively young age of BPDCN cases in our research, and sample sizes should be increased in the future to reduce statistical deviation.
In summary, our study is the first assessment about the positive rate and diagnostic efficacy of NRP-1/CD304 in various common hematological diseases with large samples. NRP-1/CD304 is only expressed in BPDCN, B-ALL and AML, but not in other common hematological diseases. This indicates that NRP-1/CD304 has no obvious diagnostic and follow-up study value in hematological diseases other than BPDCN, B-ALL and AML.