A novel zinc finger gene, ZNF465, is inappropriately expressed in acute myeloid leukaemia cells.

To increase our knowledge of leukaemia-associated antigens, especially in acute myeloid leukaemia (AML) M4, we prepared a phage display cDNA library using mRNA from the bone marrow cells of a patient with AML M4 at diagnosis. We immunoscreened 10(6) pfu with autologous sera and identified an antigen which we named GKT-AML8. The cDNA showed more than 99% similarity to a sequence on 2q21.2 and 95% sequence similarity to a sequence on 19q13.3. These genes were named ZNF465 and ZNF466, respectively, following HUGO Gene Nomenclature Committee (HGNC) guidelines. Expressed sequence tag data suggests that both genes are transcriptionally active. ZNF465 and ZNF466 encode a 5' krüppel associated box domain typical of negative regulators of gene transcription. We have confirmed the translational start site in the +1 frame in a near-Kozak sequence that produces a 102 amino acid polypeptide from ZNF465. The high level of sequence similarity between ZNF465 and ZNF466 makes their transcripts almost indistinguishable by real-time polymerase chain reaction (RT-PCR). However, GKT-AML8 showed most sequence similarity to ZNF465 and no transcript matching the 3' ZNF466 sequence could be detected in patient samples or healthy volunteers. ZNF465/466 expression was detectable in 12/13 AML and 10/14 chronic myeloid leukaemia patients' samples but not in normal donor peripheral blood (0/8) or 0/3 bone marrow samples which had been separated into CD34(+) and CD34(-) samples. The altered expression of ZNF465/466 in patients' samples and its absence in healthy donor haematopoietic samples indicate that ZNF465 is overexpressed in early myeloid disease and as such may represent a promising target for immunotherapy.

Optimised SEREX technique for the identification of leukaemia-associated antigens.

The serological analysis of antigens by recombinant expression cloning (SEREX) has been used by many laboratories to immunoscreen lambda phage cDNA libraries produced from a range of tumour cell types. We and others have found it difficult to extract an optimal quality and quantity of mRNA for the preparation of cDNA libraries which represent the genes transcribed in haematological samples. The difficulty is believed to be due to residual haem groups in the isolated RNA sample which inhibit the activity of reverse transcriptase used in the later production of cDNA. During our preparation of a cDNA library for SEREX studies, we optimised the isolation of mRNA from samples from patients with haematological malignancies. We compared the efficacy of different methods of mRNA extraction using a range of haematological sample sizes and describe the most efficient techniques to maximise mRNA yield and quality for cDNA library production. The phage library we prepared contained a range of cDNA insert sizes, including high molecular weight sequences which, following immunoscreening with autologous patient sera, led to the isolation of 17 novel antigens. Using the methodology described, we have shown SEREX to be effective for the isolation of leukaemia-associated antigens, which may act as targets for immunotherapy.