The question of whether cell proliferation is monoclonal or poly clonal is fundamental to understanding the underlying origins of hematologic malignancies. Markers of clonality are used to determine the origin of disease; to differentiate malignant from nonmalignant populations; to establish hematopoietic hierarchy, clonal evolution, and clonal remission; and to delineate steps involved in the multistep pathogenesis of hematologic malignancies.
The clonal origin of leukemias and lymphomas can be assessed by either intrinsic or extrinsic cellular markers. Intrinsic cellular markers are specific for a cell population, arising either during normal differentiation or as a part of disease process. For instance, cell surface-associated immunoglobulin (Ig) markers such as the λ or κ light chain or idiotypes and T-cell receptors (TCRs) can be useful for evaluating lymphoid malignancies. Application of IgH markers demonstrated for the first time that MM was of clonal origin. Somatic cytogenetic alterations are useful intrinsic markers for identifying abnormal clones and following disease progression. Thus the observation of identical chromosome anomalies in different cells of the same tumor is evidence of clonality. Since the discovery of the Ph in 1960 it has been well established that nonrandom, recurrent chromosomal abnormalities characterize many hematologic malignancies. The finding of the Ph in different CML-derived hematopoietic cell lineages led to the hypothesis that CML originates in a single precursor cell that has a clonal development pattern. Moreover, the presence of additional recurrent chromosomal abnormalities in the Ph-positive clone (such as trisomy 8, duplication of the Ph, or trisomy 19) not only indicates the progression of the disease to accelerated phase or blast crisis, but also demonstrates the subclonal evolution of the Ph-positive clone. Currently, disease-associated somatic genomic mutations, such as rearrangements of KMT2A (MLL), runt-related transcription factor gene (RUNX1), ETV6, PML RARA, and many others, can be identified by PCR-based assays, FISH assay, and novel aCGH and NGS, and may serve, with or without conventional cytogenetics, as intrinsic markers of disease processes.
On the other hand, extrinsic marker systems use cellular mosaicism that is completely independent of the disease being studied and is not restricted to the cell lineages. Individuals with Turner or Klinefelter syndrome are mosaic for XX or XY and monosomy X cells or XXY and XY cells, respectively. The mosaicism created by X-chromosome inactivation in females is much more widely applicable, and initially provided fundamental insights into the pathogenesis of hematologic malignancies. Original studies with X-linked glucose-6-phosphate dehydrogenase (G6PD) as a marker of clonality were based on the Lyon hypothesis, which asserts that early in embryogenesis, one X chromosome in females is inactivated in somatic cells and the activation status is stably transmitted to daughter cells during mitosis (Fig. 1). The choice of maternal versus paternal X-chromosome inactivation is random; however, once it occurs, it is maintained in all daughter cells. Random X inactivation occurs by embryonic day 6.5 around the start of gastrulation and results in a mosaic pattern that characterizes adult females. Therefore an adult female is a mosaic for two-cell populations, one expressing genes from an active X chromo some and the other expressing genes from the inactive X chromosome. Incidentally, mammalian X-chromosome inactivation is a mechanism that equalizes the dosage of X-linked genes between sexes. Although the exact mechanism of X-chromosome inactivation remains to be elucidated, the process of X inactivation starts with methylation of CpG islands. The inactivation process is believed to occur before differentiation of the embryonic stem cell into various cell lineages. Hematopoietic cells do not originate from a single embryonic stem cell but from several hematopoietic stem cells, thereby allowing for mosaic expression from both X chromosomes.
Fig1. X-CHROMOSOME–LINKED ENZYME GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) AS A MARKER TO INVESTIGATE CLONAL DEVELOPMENT OF HUMAN HEMATOPOIETIC DISORDERS. Early in embryogenesis, regions of all but one X chromosome are inactivated in each cell containing two or more X chromosomes. The choice of maternal versus paternal X chromosome for inactivation is random. Once the inactivation occurs, it is fixed and is stably transmitted to daughter cells during mitosis (Lyon hypothesis). Females who are heterozygous for the common B type and the less frequent A type, G6PD (localized on Xq27), are mosaic. This cellular mosaicism is used to study monoclonal versus polyclonal cell proliferation and development of malignant hematopoietic diseases. (Courtesy Dr. W Raskind, University of Washington, Seattle.)
The observation that human females were heterozygous for the G6PD variant A and A− and that two mosaic cell populations may be distinguishable by electrophoretic mobility was reported in the 1960s. The X-inactivation G6PD mosaic system was then applied to the study of clonality in human tumors (uterine leiomyomas) in 1964 by Gartler and Linden. In females who were heterozygous for the G6PD polymorphism and had malignant hematologic disorders such as CML, the finding of a single G6PD type in marrow or blood cells and both the A and B type G6PD in tissues not involved by the malignant process first demonstrated that CML was of clonal origin and provided evidence that the malignant transformation occurred at the level of a stem cell common to most hematopoietic cell lineages. Additional studies with heterozygous G6PD females who had CML demonstrated that some CML-derived B lymphocytes had a single G6PD type, but these clonal cells were Ph-negative; thus leukemic transformation might predate development of the chromosomal abnormality. This observation provided evidence that CML has a multistep pathogenesis. Application of this approach to hematologic malignancies demonstrated the clonal and stem cell origin for AML, ALL, Ph-negative myeloproliferative neoplasm (MPN), MDS, and CLL.2 Studies using G6PD were particularly useful in the investigation of red blood cells and platelets in hematologic malignancies because the absence of nuclei in these cells did not allow them to be studied with cytogenetics or DNA analysis. Although it is now considered common knowledge that hematologic malignancies are characterized by clonal development, this concept was largely developed by Dr Phillip Fialkow.
Despite the importance of the G6PD approach, it is limited by the rarity of females who are heterozygous for the G6PD isoenzymes. An alternative and more extensive DNA-based X-chromosome clonal assay used common polymorphic markers that are caused by changes in DNA methylation patterns that accompany inactivation of the X chromosome. These X-linked loci such as phosphoglycerine kinase, hypoxanthine phosphoribosyltransferase, DXS25 (M27β), and human androgen receptor (HUMARA), have been extensively used in assessment of clonality, and can be used to identify clonal cell populations in virtually all females. DNA-based marker systems rely on a sequence polymorphism that has adjacent differences in methylation on the active and inactive X chromosomes. The inactive X chromo some is more highly methylated than its active homologue, but this is only true for certain regions of genes as 10% to 20% of X-linked genes escape inactivation and can be found both in clusters and in isolation. The most widely used HUMARA assay appears to maintain stringent methylation differences. The number of CAG tandem repeats differentiates the maternal from the paternal X chromosome.
The utility of the DNA-based X-chromosome clonal assay is limited to females younger than 60 years because they usually have 1:1 distribution of two-mosaic–cell population. A ratio greater than 3:1 is found in women older than 60 years, probably as a result of stem cell kinetics influenced by X-linked genetic factors. When the ratio of two cell populations is greater than 3:1, this phenomenon is called a skewed X-inactivation pattern. With the HUMARA assay, acquired unequal or skewed X-chromosome inactivation (excessive lyonization) is found in 35% to 40% of women older than 60 years. Thus X-chromosome based clonality studies must incorporate age-matched controls. More recently acquired somatic mutations using NGS have been useful in documenting clonal hematologic malignancies.
