Leukemia (2000) 14, 935–940  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu BIO-TECHNICAL METHODS SECTION (BTS) BTS Leukemia Detection of methylthioadenosine phosphorylase (MTAP) and p16 gene deletion in T cell acute lymphoblastic leukemia by real-time quantitative PCR assay TJ M’soka 1 , J Nishioka 2 , A Taga 2 , K Kato 2 , H Kawasaki 1 , Y Yamada 3 , A Yu 4 , Y Komada 1 and T Nobori 5 1 Department of Pediatrics, 2 Central Clinical Laboratories, 5 Department of Laboratory Medicine, Mie University Hospital, Tsu, Mie; 3 Department of Laboratory Medicine, Nagasaki University School of Medicine, Nagasaki, Japan; and 4 Department of Pediatrics, University of California at San Diego, California, USA Methylthioadenosine phosphorylase (MTAP) deficiency in tumors can be therapeutically exploited for selective therapy. Many tumors lacking MTAP have been found to homozygously delete the chromosome 9p region containing the p16 tumor suppressor gene. Several methods have been used to detect chromosome 9p deletions in primary tumors. However, the accurate diagnosis of chromosome 9p deletions has been ham- pered by the presence of contaminating normal cells. In search of an accurate and sensitive diagnostic method, we have developed the real-time polymerase chain reaction assay using the TaqMan chemistry for quantitative detection of MTAP and p16 gene deletions. The assay’s feasibility was tested with per- ipheral blood leukocytes (PBL) from 29 patients with adult T cell leukemia (ATL) previously analyzed with Southern blot analysis and validated on 39 PBL or bone marrow samples from childhood T cell acute lymphoblastic leukemia (T-ALL). Homo- zygous deletions of MTAP and p16 genes were detected respectively in six (20.7%) and eight (27.6%) of 29 ATL samples and in 15 (38.5%) and 23 (59%) of 39 T-ALL samples. The results correlated well with those of Southern blot analysis. It is of sig- nificance that the newly developed method can successfully detect homozygous deletions of these genes in samples con- taining as low as 33% blast cells. This rapid and sensitive method may be useful in searching for candidates for selective therapy targeting MTAP deficiency. Leukemia (2000) 14, 935– 940. Keywords: MTAP deficiency; chromosome 9p deletions; real-time quantitative PCR; ATL; T-ALL Introduction Methylthioadenosine phosphorylase (MTAP) deficiency in tumor can be therapeutically exploited for selective ther- apy. 1–3 MTAP, the housekeeping enzyme, plays a major role in the salvage of both adenine and methionine with 59-deoxy- 59-methylthioadenosine (MTA) as substrate. 4,5 This salvage pathway is the major source of adenine in human cells. 6 While MTAP deficiency has been demonstrated in many tumor cell lines and primary tumors such as leukemias, mela- nomas, brain tumors and non-small cell lung cancers, 7–11 all normal cells including erythrocytes and hematopoietic stem/ progenitor cells contain the enzyme in abundance. 12,13 We Correspondence: T Nobori, Department of Laboratory Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; Fax: (81) 59-231-5250 Received 9 December 1999; accepted 19 January 2000 and others have shown that MTAP deficiency in cancer offers a specific molecular target for selective therapy in in vitro studies. 2,14–16 Cells deficient in MTAP are unable to utilize MTA, the natural enzyme substrate, or a substitute such as 59-deoxyadenosine to synthesize methionine and/or adenine nucleotides. 1,7 By combining with MTA or 59-deoxyadenosine to rescue normal cells, bacterial L-methioninase or purine de novo synthesis inhibitor L-alanosine can selectively kill MTAP deficient tumor cells in vitro. 2,3,14 Recently it was reported that MTAP deficient T cell acute lymphoblastic leukemia (T-ALL) cells were selectively killed with L-alanosine and that normal lymphocytes were rescued with 59-deoxyadenosine. 1 The MTAP gene resides on chromosome 9p about 100 kb telomeric to the tumor suppressor genes p16 and p15 which encode negative regulators of the cell cycle. 17–19 The deletion of p16 has been demonstrated in many cancers and associated with carcinogenesis as well as disease progression in some cancers. 20–23 The MTAP gene is frequently co-deleted with the p16 gene in adult T cell leukemia (ATL) and childhood T-ALL. 3,24 ATL, clinically a heterogeneous disease, responds poorly to therapy. 25 The deletion of p16 gene predicts the shortened survival in ATL and plays a key role in the progression of the disease from chronic to acute form. 26,27 Although no such relationship existed at diagnosis, p16 deletion was associated with the shorter survival in patients with relapsed T-ALL. 28 Since chromosome 9p deletions in ATL and T-ALL predict prognosis, a precise and accurate diagnostic method for detection of such gene deletions is needed. We and other investigators have previously reported the use of various methods to determine the gene status at chromosome 9p, including MTAP enzyme assay, Western blot analysis, fluor- escence in situ hybridization (FISH), end-point quantitative PCR and Southern blot analysis. 2,3,10,29 The accurate diagnosis of chromosome 9p deletions has been hampered by the pres- ence of contaminating normal cells in primary samples. In search of a sensitive and accurate method, we have developed a real-time quantitative PCR (RQ-PCR) assay based on the TaqMan chemistry (Perkin Elmer-Applied Biosystems ABI Prism 7700 sequence detection system) to evaluate MTAP and p16 gene status in samples from patients with ATL and T-ALL. This method for nucleic acid quantification is fully automated, rapid and sensitive and eliminates the need for post-PCR processing, a potential source of carryover contamination.