Is Hematopoiesis Clonal in Healthy Elderly Females?

Clonality studies can establish the single cell origin of tumors and differentiate nonmalignant from malignant states. Detection of clonal cells may be genotype-based relying on somatic mutations to mark the clonal population (e.g. 9q+:22q– translocation in CML), or phenotype-based, where the clonal population is identified by expression of surrogate genes which facilitate tracking the clonal process. Methods for determining phenotypic clonality rely on the principle of X chromosome inactivation (XCIP), unique to women, and are based on differentiating transcriptionally active from inactive X-chromosomal genes. Detection of the polymorphic state of genes subjected to inactivation may be done by either: discrimination of DNA methylation status, detection of mRNA transcripts, or polymorphic isozyme protein products. Extreme skewing of X-chromosome allelic usage by methylation-based clonality assay has been reported in ∼30% of healthy elderly females precluding clonality studies in this population. In contrast, by X-chromosome quantitative transcriptional clonality assay (TCA), we previously reported a normal skewing range based on our determination of random X-chromosome inactivation in 8 progenitors of pluripotent hematopoietic stem cells at the time of inactivation during the blastocyst stage of development (J Exp Med, 183:748, 1996). Moreover, we have not observed clonal XCIP in TCA studies involving over 200 healthy heterozygous females, indicating the rarity of this phenomenon. However, we did not systematically study females >65 years old. Furthermore, our TCA protocol was laborious, technically demanding and required significant amounts of highly radioactive isotopes. In addition, due to susceptibility of DNA methylation to environmental factors we decided to re-investigate the issue of clonality in older females using a novel quantitative real time PCR assay based on a unique primer design, we previously reported for the JAK2V617F mutation (Exp Hematol. 2007;35(1):32–8). Females >65 years of age with no history of malignant disorders, unexplained anemia, or autoimmune disorders were recruited for our IRB approved study. Genomic DNA and total RNA was isolated from peripheral blood granulocytes, reticulocytes, and platelets where applicable. Clonality studies were performed using BTK, FHL1, IDS, G6PD, and MPP1 exonic polymorphisms (∼95% females are informative for at least one marker; Blood101:3294–301, 2003). Genomic DNA was used for genotyping exonic polymorphisms by TaqMan based allelic-discrimination assays. Thirteen elderly females (age range 65–92, mean 75.5, median 75) and 5 younger females (age range 30–40, median 36, mean 35), heterozygous for one or more markers, were identified. TCA was performed using total RNA on markers found to be informative. Neither clonal XCIP, nor extreme skewing XCIP was noted in any of the study subjects. Based on reported data, ∼30% elderly women were found to have extremely skewed XCIP; hence, we expected to find 4/13 elderly women with clonal XCIP in our study group. Statistical analysis, using an exact binomial test, indicates a low probability of false positive results by our assay (p=0.014, exact 95% CI [0,0.22]). In conclusion, hematopoiesis is not clonal in healthy elderly females. #equal contribution.