Phosphoinositide phospholipase Cbeta1 (PI-PLCbeta1) gene in myelodysplastic syndromes and cytogenetically normal acute myeloid leukemia: not a deletion, but increased PI-PLCbeta1 expression is an independent prognostic factor.

TO THE EDITOR: Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and propensity to myeloid leukemic transformation. Initially, the French-AmericanBritish and WHO classifications distinguished several subtypes. The International Prognostic Scoring System considered the number of cytopenias, the karyotype, and the percentage of bone marrow blasts. Prognostic parameters for survival and progression to acute myeloid leukemia (AML) have been proposed, including circulating blasts, lactate dehydrogenase serum levels, and RBC transfusion requirement. However, prognostic factors for evolution into AML are lacking. Therefore, the observation of a specific monoallelic deletion in the phosphoinositide phospholipase C 1 (PI-PLC 1) gene in 35 (43.75%) of 80 MDS patient samples and the prognostic impact of these deletions on evolution from MDS to AML described by Follo et al, may represent an important advance. The PI-PLC 1 deletion status was an independent prognostic marker for evolution into AML and worse clinical outcome. Furthermore, Follo et al demonstrated that patients with this specific deletion had greatly reduced PI-PLC 1 mRNA transcript levels. Since these findings have the potential to change routine cytogenetic investigation for MDS and suggest PI-PLC 1 to be a strong candidate as a potential target for novel therapeutic approaches, we performed fluorescent in situ hybridization (FISH) analysis on 32 patients with MDS and 20 patients with cytogenetically normal (CN) AML to identify this deletion. FISH investigations were performed on interphase nuclei and metaphases as described earlier and the same P1 artificial chromosome (PAC) clone described by Follo et al (PAC clone RP5-881E24; de Jong PAC library, Childrens Hospital Oakland, Oakland, CA) was used to generate probes as described in detail previously. In each case, at least 200 interphase nuclei and 10 metaphases were analyzed. BAC clones for the subtelomeric region 20p were used as a control (RP11-147L21 and RP11-530N10 by BACPAC resources). In order to minimize the risk of false-positive results from FISH analysis (eg, due to insufficient hybridization), it is necessary to determine a cutoff level for each probe tested. Through analyses of at least 1,000 cells from four healthy donors, the cutoff level for our probe combination was determined to be 9% (standard deviation 3; data not shown). Results with a signal constellation for a PI-PLC 1 deletion below this were considered negative. Our series included 36 men and 16 women, age 31 to 79 years. Characteristics of the patient cohort are shown in Table 1. Surprisingly, we did not identify a monoallelic deletion in the PI-PLC 1 gene region in any of the 52 bone marrow samples tested (Fig 1). These results are in contradiction with those of Follo et al as they observed a monoallelic deletion of the PI-PLC 1 gene in 35 (43.75%) of 80 patients with MDS. In an earlier report, some of the same investigators also described PI-PLC 1 monoallelic deletions in two (33.3%) of six AML cases. While although the reason for the discrepancy between our results and those of Follo et al is not immediately clear, one explanation might be that our cutoff level for assigning PI-PLC 1 deletion was 9% whereas Follo et al did not mention a cutoff level for their analyses. In general, an accepted practice to reduce the risk of false-positive results from FISH analysis is that each laboratory performing FISH analyses must determine appropriate cutoff levels for each probe tested. Our results are in concordance with the report by Herens et al, who found no PI-PLC 1 monoallelic deletions in samples from 23 with MDS and seven patients with AML. Thus, the incidence of PI-PLC 1 monoallelic deletions in patients with MDS and AML remains to be confirmed. Interestingly, Follo et al demonstrated that patients with a specific PI-PLC 1 deletion had greatly reduced PI-PLC 1 expression levels. In order to investigate the hypothesis that PI-PLC 1 transcript levels have an impact on clinical outcome, we quantified PI-PLC 1 mRNA transcript levels on a large cohort of 245 patients with AML treated on two multicenter treatment trials AML SHG 0199 (Clinical Trials Identifier NCT00209833, June 1999 to September 2004, N 164) or AML SHG 0295 (February 1995 to May 1999, N 81). Detailed information on treatment schedules and patient characteristics were described earlier. Reverse transcriptase polymerase chain reaction was accomplished on a StepOne Plus real-time PCR system (Applied Biosystems, Darmstadt, Germany) using a commercially available TaqMan Gene Expression Assay (PI-PLC 1 probe: Hs00248563_m1; Applied Biosystems) and the ABL FusionQuant Standards Kit (Ipsogen, Marseille, France) as an endogenous control. Expression levels were quantified according to the manufacturer’s instructions. All reactions were quantified in duplicate and data was analyzed using the StepOne Plus Software version 2.0 (Applied Biosystems). Interestingly, the 122 patients with AML with high PI-PLC 1 mRNA expression levels (defined as expression above the median expression level) had a significantly shorter OS (P .032; Fig 2) compared with the 123 patients with AML with low PI-PLC 1 transcript levels. No difference for relapse-free survival (P .305; Fig 2) was observed. In multivariate analysis for OS, PI-PLC 1 expression status independently predicted an adverse prognosis (P .009; HR, 1.658; 95% CI, 1.132 to 2.427) when considered together with NPM1/FLT3 mutation status, CEBPA mutation status, WT1 SNP rs16754, WT1 expression status, age, WBC count, and platelet count (Table 2). JOURNAL OF CLINICAL ONCOLOGY C O R R E S P O N D E N C E VOLUME 28 NUMBER 22 AUGUST 1 2010