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Chapter 56 Conventional and Molecular Cytogenomic Basis of Hematologic Malignancies 783

or skewed X-chromosome inactivation (excessive lyonization) is and a gain of 22q11.1–q11.23 region, whereas twin B had deletions
found in 35% to 40% of women older than 60 years. Thus of EBF1 and IKZ1. These genotyping results provide direct evidence
X-chromosome–based clonality studies must incorporate age-matched that rearrangements additional to BCR-ABL1 are obviously postnatal
controls. Despite the enormous contribution of clonality assays to the in origin, they represent a subclonal evolution, and BCR-ABL1 by
understanding of disease processes, they are usually performed in itself is not sufficient for development of Ph-positive ALL.
research investigations and are rarely used as diagnostic tools. These mutation-driven natural history studies provide evidence
More recently acquired somatic mutations are detected using for sequential multistep pathogenesis with sequential accumulation
NGS, also known as high-throughput sequencing, the catch-all term of genetic changes, which may be linear through clonal succession as
used to describe a number of different modern sequencing technolo- originally proposed by Peter Nowell in 1976, but as Greaves indicated,
gies whereby millions of small fragments of DNA can be sequenced clonal evolution in most leukemia is rather complex and may repre-
at the same time, creating a massive pool of data. This pool of data sent also a branching structure, as predicted by Darwin. 2
can reach gigabites in size, which is the equivalent of 1 billion
(1,000,000,000) base pairs of DNA.
Although NGS makes genome sequences handy, the data analysis EARLY MUTATIONS IN LEUKEMOGENESIS AND AGE-
and biologic explanations are still the bottle-neck in understanding RELATED CLONAL HEMATOPOIESIS
leukemic genomes.
As indicated earlier and throughout the chapter, the progress of
high-throughput sequencing has provided novel observations in the
In Utero Mutations and Clonal Origin current understanding of the initial mutations in premalignant stem
cells and the clonal development through the accumulation of genetic
The observation that monozygotic twins share identical but nonconsti- changes. For example, the evolving concept of the clonal origin of
tutive and clone-specific fusion gene sequences (e.g., ETV6-RUNX1) AML involves the mutation in the gene encoding DNA methyltrans-
3
in pediatric ALL provided the first unambiguous evidence (in 2003) ferase 3A, DNMT3. Approximately 22% of patients with AML have
that genetic lesions, generated by chromosomal translocation, arise in a mutation in DNMT3 gene, localized on the short arms of human
utero. These sophisticated series of studies initiated by Mel Greaves chromosome 2, 2p23. However, mutations of this gene are also
and his colleagues at the Institute for Cancer Research in London, described in other myeloid malignancies as well as in T cell leukemia/
have contributed for the past 13 years to a wealth of knowledge about lymphoma. Deep sequencing of patients with AML showed that
founder mutations, subclonal development, clonal origin, and the DNMT3 mutations are typically found at higher frequencies than
evolution of disease. The initiating lesion and premalignant clone other accompanied mutations in AML, such as NPM1, FLT3 or
is shared by the twins as a consequence of intraplacental vascular others, suggesting that they were among the first to arise. Two studies
anastomoses and blood cell chimerism. The twin data are endorsed demonstrated that patients with AML and DNMT3 mutations had
by backtracking of prenatal-initiating genetic lesions in the archived the same mutation in T and B lymphocytes, indicating that in these
blood spots, or Guthrie cards, of patients with ALL. These data were patients the mutation had occurred in a primordial cell giving rise to
interpreted to suggest that ETV6-RUNX1 is likely to be a critical all hematopoietic lineages, which may represent a “founder” clone
initiating lesion for ETV6-RUNX1–positive ALL. However, such population from which the AML leukemic population expands.
fusions are detectable in cord blood from newborn infants at rates Co-occurrence of DNMT3 mutations in patients with chromosomal
approximately 100-fold higher than the incidence of ALL, suggest- abnormalities such as t(15;17), inv(16), and t(8;21) (see later in the
ing an obligatory requirement for additional mutations in leukemia chapter) have not been described. In sharp contrast, approximately
development. Over a period of 10 years these results were confirmed 60% of patients with DNMT3 mutation also carry an NPM1 muta-
and in utero origin of MLL, BCR-ABL1, and RUNX1-RUNXT1 tion whereas only 13% of the patients with the wild type DNMT3
fusion rearrangements were documented, providing direct evidence carry NPM1 mutations. These patterns imply a temporal acquisition
for a prenatal origin of many childhood leukemias. of mutations in AML. Collectively these and other observations
Results from genome sequencing of ETV6-RUNX1 fusion region indicate that the DNMT3 mutation is a primary mutation in prema-
suggests it arises as a consequence of nonhomologous end-joining in lignant stem cell whereas NPM1 and FLT3 are common secondary
the pro-B cell stage with possible self-renewal capacity to downstream mutations in more differentiated cells that lead to an acute phase of
B-cell precursors. Additional evidence was provided by screening for disease, and combined, they may represent a distinct AML entity. It
trisomies in stored cord blood and the data indicated that ~6% of has been further documented that DNMT3 mutant hematopoietic
+
+
enriched CD34 /CD19 B lineage progenitors carry trisomies fre- stem cells and their differentiated progeny persisted in the peripheral
quently seen in hyperdiploid childhood ALL. With novel SNP and blood of these patients even when their AML is in remission follow-
other technologies, it has become apparent that ALL has multiple ing chemotherapy, indicating that at least some of these preleukemic
genome copy number variations (CNVs), mostly deletions and these ancestral cells are resistant to treatment. These studies have significant
CNVs are distinctive between a pair of twins, indicating a secondary, implications for the development of targeted therapies.
postnatal origin. 1 Clonal mosaicism for large chromosomal anomalies (duplications,
When genotyping sequencing combined with other molecular deletions, and uniparental disomy [UPD]) using SNP microarray
technologies of five pairs of monozygotic twins with concordant data from 50,000 subjects in the GENEVA study determined that
ETV6-RUNX1-positive ALL, Greaves and his colleagues demon- clonal hematopoiesis is infrequent (<0.5%) from birth until 50 years
strated that all recurrent CNVs (32 in total) were different within of age after its frequency rapidly increases to 2% to 3% in the elderly.
twin pairs providing strong evidence that they are probably secondary It has been estimated that individuals with clonal hematopoiesis have
mutations and postnatal in origin in both twins as well as in nontwins 10-fold higher risk of a subsequent hematologic malignancy. These
with ALL. Another two twin pairs who shared a monochromaric age-related mutations were confirmed by analysis of mutation acqui-
placenta and had Ph-positive ALL, also studied by Greaves and his sition in hematopoietic stem cells, over time, through whole-exome
colleagues, provided confirmation for the previous observation that sequencing of single hematopoietic stem cell-derived colonies, which
BCR-ABL1 is not sufficient to cause Ph- positive leukemia. Twin A showed that the total number of mutations in healthy individual stem
presented with ALL at age 3.8 years and twin B presented at age 4.1 cells increases with age. Moreover, sequencing of multiple elderly
years. Both had an identical BCR-ABL1 fusion transcript and both women provided evidence of clonal hematopoiesis based on X inac-
received an allogeneic stem cell transplantation from the same human tivation pattern and recurrent somatic mutation in TET2 gene.
leukocyte antigen (HLA) identical sibling donor but 7 months later Subsequent analysis of 182 additional elderly women with clonal
twin B died whereas twin A is in good health 8 years following the hematopoiesis determined that more than 5% of these individuals
transplantation. SNP analysis revealed that twin A had a subclone had mutations in TET2. Most recent reports by Ebbert and his
with trisomies for chromosomes 4, 6, 9, 14, 17, and X, tetrasomy 21, colleagues in USA on 17,182 persons and by Cross and his colleagues