NM23-H2 Is a Tumor Associated Antigen (TAA) in Chronic Myeloid Leukemia (CML).

Introduction: The human Nm23 family consists of 8 genes, termed Nm23-H1 through -H8. H1 and H2 are 88% identical and localized to chromosome 17. Both Nm23-H1 and -H2 display nucleotide-diphosphate kinase (NDPk) activity responsible for balancing NTP levels by transferring gamma-phosphates between nucleotides, and individually demonstrate a variety of other activities including DNA binding, transcriptional regulation and apoptotic cleavage of DNA. A variety of studies imply a role for Nm23 proteins in development and in a range of cancers, in which level of expression rather than mutation correlates to prognosis. Specifically, a mutual transcriptional activation between the nm23-H2 and c-myc genes has been implicated in transformation. Our group described the identification of Nm23-H2 as a candidate tumor-associated antigen in a case of CML and demonstrated the presence of Nm23-H2 reactive T cells 5 years after transplantation. Here, we compare the expression of Nm23-H2 and H1 proteins in leukaemic patients and healthy donors.

Materials and Methods: PBMC were collected from 10 CML patients at initial diagnosis, from 4 Glivec-resistant CML patients, from 12 AML patients at diagnosis and from 5 healthy donors. PBMC, CD3⁺, CD14⁺, CD33⁺ and CD34⁺ populations were stained, fixed and sorted by FACS onto microscope slides. Immunocytochemical analysis was performed with Nm23-H1 (Novacastra), Nm23-H2 (Santa Cruz) and β-Actin (Sigma) specific antibodies. Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss).

Results: PBMC from 9 out of 10 untreated CML patients (90%) showed a strong Nm23-H2 staining. Fractionation of the PBMC population revealed that CD14⁺, CD33⁺ and CD34⁺ but not CD3⁺ cells are Nm23-H2 positive in the majority of these patients. Nm23-H2 was also detected in the PBMC of 3 out of 4 Glivec-resistant CML patients. In most cases, Nm23-H2 co-localized with β-actin. Under the same staining conditions, PBMC and sorted subpopulations from 11 of 12 AML patients (92%) and from all healthy donors were Nm23-H2 negative, while very weak staining was detected in PBMC of one AML patient. Nm23-H1 protein was clearly detectable in a number of cell lines (HL-60, U-937, K562 and T47D), but was very weak or absent in PBMC from CML patients and healthy donors.

Conclusion: Taken together, our results show that those myeloid populations functionally affected by the bcr/abl mutation specifically over express Nm23-H2 but not Nm23-H1 protein in 9 out of 10 cases of CML. While the molecular basis of this expression pattern and the possible involvement of c-Myc remains to be resolved, these results support the proposal that immune responses to Nm23-H2 may mediate GvL effects and that peptides derived from Nm23-H2 might therefore be attractive targets for specific immunotherapy in CML patients.