Conventional and Molecular Cytogenomic Basis of Hematologic Malignancies

Cytogenetic Analysis

Cells arrested in metaphase are obtained by exposing marrow cells sequentially to mitotic inhibitors, hypotonic potassium chloride, and fixative. Chromosomes obtained from leukemic marrow are then subjected to trypsin-Giemsa banding ( Fig. 57.2 ). The criteria used to define clonal abnormalities are listed in Table 57.1 and described in the International System for Human Cytogenetic Nomenclature, 2021. Table 57.2 describes the optimal logarithm and evolving new testing strategies. These technologies are currently either stand alone or are used in combination.

Fluorescence in Situ Hybridization Methods

Fluorescence in situ hybridization (FISH) is a molecular method that allows detection of the number, size, and location of DNA and RNA segments within individual cells in a tissue sample. It is based on the ability of single-stranded DNA to anneal to complementary DNA. In hematologic disorders, the target DNA is the marrow or peripheral blood DNA present in interphase cells or the DNA of metaphase chromosomes that is fixed on a microscope slide.

Fig. 57.3 shows four types of FISH probes that are used alone or in combination to determine both numeric and structural rearrangements: (a) centromere enumeration probes, (b) whole chromosome painting probes, and (c) subtelomeric probes, while Fig. 57.4 shows the four FISH probe strategies used in probe design for detection of chromosomal translocations in hematologic malignancies. The first application of FISH technology for detection of chromosomal translocations in hematologic malignancies occurred when the BCR-ABL1 hybrid gene was identified using two-color FISH in interphase cells as well as in metaphase marrow-derived CML cells. In the standard strategy for interphase evaluation of chromosomal translocation, a DNA probe comprising sequences mapped proximal to the breakpoint in one of the chromosomes involved in reciprocal translocations is combined with a differentially labeled DNA probe that includes sequences mapped distal to the breakpoint in the other chromosome. Nuclei positive for the translocation display one dual-color fusion signal, representing one of the derivative chromosomes generated by the translocation, and two single-color signals, one for each of the normal alleles. This standard FISH strategy has been used for detection of translocations in hematologic disorders at diagnosis.

One of the most significant advances in diagnostic leukemia cytogenetics has been the application of interphase FISH. Interphase cytogenetics is the term used to describe detection of chromosomal abnormalities in non-dividing, interphase nuclei ( Fig. 57.5 ). Five aspects of interphase FISH are particularly useful: (1) Interphase cytogenetics allows screening of a large number of cells. This permits investigation of hematologic malignancies with a low mitotic yield, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). (2) Interphase FISH permits detection of chromosomal rearrangements in peripheral blood samples, thus obviating the need for marrow aspiration. For instance, in CML, which rarely yields a large number of dividing cells in peripheral blood, conventional cytogenetics usually is uninformative. However, detection of BCR-ABL1 , the molecular equivalent of the Philadelphia chromosome (Ph), in peripheral blood using interphase FISH provides reliable, fast, quantitative results. (3) Interphase FISH offers a quantitative assay for monitoring disease progression or detection of minimal residual disease (MRD) following chemotherapy or hematopoietic stem cell transplantation (HSCT). (4) Use of specific probe sets allows detection of specific disease-associated abnormalities such as t(8;21), which denotes the M2 subtype of acute myeloid leukemia (AML), or t(15;17), which is associated with acute promyelocytic leukemia (APL), within 4 hours, allowing for timely and appropriate therapy. (5) FISH nomenclature is described in the International System for Human Cytogenetic Nomenclature.

Multicolor karyotyping permits examination of the entire genome in a single analysis ( Figs. 57.1 and 57.6 ). In 1996 it became possible to identify 24 different human chromosomes (22 autosomes and the X and Y sex chromosomes), each with a unique color, with the help of fluorochrome-specific optical filters. This method is called multicolor FISH (M-FISH) . When interferometer-based spectral imaging is used, the method is called spectral karyotyping . The starting point in both methodologies is the use of whole chromosome painting probes for each chromosome. Thus each chromosome is labeled with a different combination of fluorescent dyes and images are sequentially obtained using five different fluorochrome-specific optical filters. A computer program combines the data and displays each chromosome as if it were stained with a distinct color. Spectral karyotyping is based on the use of an interferometer (used by astronomers to measure the light spectra of distant stars) to determine the full spectrum of light emitted by each stained chromosome. A computer program then displays all the chromosomes simultaneously, each with its own unique color. These methods are applied with increasing frequency to resolve complex karyotypes and to resolve the origin of the marker chromosome.

Comparative Genomic Hybridization and Next Generation Sequencing

Another powerful method used for identifying the location of chromosomal gains, losses, deletions, or amplifications, without prior knowledge of the chromosomal target that may be altered, is comparative genomic hybridization (CGH) (see Fig. 57.1 ). Briefly, isolated DNA from a patient’s marrow or tumor tissue is labeled with a one-color fluorochrome (e.g., red), whereas DNA isolated from normal control tissue is labeled with a different color (e.g., green). These differently labeled DNAs are hybridized against each other in a competitive hybridization reaction onto normal metaphase spreads. Computer-assisted image analysis detects colors generated after hybridization, which indicate equal hybridization, relative excess, or deficiency of the target DNA (relative to control). The ratio of color intensity provides a “copy number” karyotype.

A particularly useful investigational approach is a “microchip array” in which labeled DNA or RNA from the sample of interest is hybridized with defined target sequences immobilized on a solid support system. This method can screen for genes that are gained/amplified or deleted from the genome on a large scale or, in the case of RNA, to learn whether such genes are expressed at a particular stage of disease. The first example of successful application of the RNA microchip array technique was the discrimination of AML from acute lymphoblastic leukemia (ALL) based solely on gene expression. As shown in Fig. 57.1 , in the array CGH (aCGH) procedure, large-insert genomic clones, oligonucleotides, or single-nucleotide polymorphisms (SNPs) have replaced metaphase chromosomes used in routine CGH. Array CGH is a higher-resolution CGH technology of approximately 5 to 50 kb and provides diagnostic information for diseases associated with DNA dosage. It can also be used to discover previously unexpected sites of altered gene dosage associated with specific hematologic malignancies. The concept of obtaining gene copy number from multiple genome locations in a single measurement has been used to characterize numerous hematologic malignancies over the last 17 years, and its clinical utility is demonstrated throughout this chapter. Nowadays, SNP arrays are also used to genotype a few hundred to millions of SNPs in order to detect rare and common genomic rearrangements (see Fig. 57.1 ). These arrays require hybridization of only the test sample onto the array, unlike aCGH, which relies on co-hybridization of test and reference DNA. At this time aCGH+SNP arrays are used together in one study and they provide exon-level resolution of genomic changes.

The most advanced genomic technologies currently known is next generation sequencing ( Chapter 3 ) or NGS (see Fig. 57.1 ). These approaches use a range of techniques that enable sequencing of hundreds of thousands of nucleic acids simultaneously. In order of complexity, these approaches include sequencing of gene panels, exome sequencing (protein-coding genes), transcriptome (expressed RNA), and sequencing the whole-genome sequencing (WGS). Implementation of these techniques requires several arrays of several hundred thousand sequencing templates in parallel generating up to several hundred million short reads of DNA sequence per lane. The basic principle of NGS involves a process of DNA fragmentation, adapter ligation, and immobilization of the fragments via the adapters to create libraries. The libraries then undergo a process of amplification, generating multiple copies of each DNA fragment which are then sequenced in parallel by a fluorescence- or chemiluminescence-based method, yielding billions of short sequence reads ( Chapter 3 ). These reads are then aligned to the human reference genome and highly efficient algorithms are used to map these complex genomes. Because of its cost effectiveness and enormous sequencing capacity, NGS is revolutionizing hematologic malignancy research by facilitating the discovery of disease-initiating mutations, identifying novel drug targets, and allowing for the first time personalized treatment strategies for hematologic malignancies. Exome sequencing is a relatively inexpensive approach to identify protein-coding mutations, but is incapable of identifying structural genetic rearrangements, deletions, and insertions of DNA, a hallmark of many hematologic malignancies. Exome sequencing is performed at a depth of 100- to 200-fold coverage of the haploid genome, which enables detection of mutations present in leukemic subclones, which is important in the study of relapse. Transcriptome sequencing involves sequencing of genes actively expressed and can be adjusted to selectively study RNA transcripts that encode proteins (mRNA), transcripts regardless of coding potential (RNA), or a variety of small and noncoding RNA transcripts. RNA sequencing is a highly informative approach which enables identification of chromosomal rearrangements that result in the expression of chimeric fusion genes and sequence mutation detection. WGS usually involves sequencing leukemic and nonleukemic DNA from an individual. Although it is the most comprehensive modality, WGS may not identify all genetic alterations caused by variations in sequence coverage and difficulties in sequencing complex and GC-rich regions of the genome (including gene promoters). These modern cytogenomic methods have increased the resolution at which chromosomal and gene rearrangements can be identified. Conventional cytogenetics, FISH, and aCGH+SNP along with NGS are complementary. Each has its own pros and cons in investigating genomic rearrangements of malignant cells.

Although conventional cytogenetics is the comprehensive study of all chromosomes, it requires a large number of dividing cells, which, in some diseases, such as myelofibrosis, is difficult to obtain (see Table 57.2 ). Furthermore, many small deletions or structural rearrangements are beyond the microscopic level of detection. FISH should be used in conjunction with conventional cytogenetics with both interphase and metaphase cells. It is a more sensitive method and detects rearrangements smaller than 1 kb. The main disadvantage of interphase FISH is that it cannot be used unless a known abnormality is suspected. When the abnormality is known, interphase FISH identifies the clonal aberration at the single-cell level. aCGH+SNP provides genome-wide higher resolution analysis and important information concerning genomic changes in patients with a normal karyotype as well as acquired loss of heterozygosity, and chromothripsis (chromosome shattering), which are important prognostic and predictive tools. With the introduction of high throughput genomic technologies such as NGS, FISH-based chromosome level detection has gradually changed focus to genome-wide detection of single nucleotide and copy number variants that are common in leukemia. It is evident that identification of chromosomal aberrations by molecular cytogenomic techniques is important in detecting novel chromosomal rearrangements and genes involved in leukemogenesis. Understanding the basis of these techniques and their application is critical in the accurate diagnosis of hematologic malignancies. Table 57.2 describes the optimal algorithm and evolving new testing strategies. New technologies, capable of simultaneously detecting copy number changes, structural variants, and mutations, are expected to be used in future in a diagnostic setting.

Genome sequencing has rapidly become routine practice. However, the intimate relationship between DNA sequencing, chromosome structure, and the position of chromosomes in the nucleus is not fully understood. The term “ chromosomics ” was recently proposed in order to integrate the original definition of cytogenetics (chromosomes and cytology) with genomics (gene content, structure and function) to ensure a closer integration of these fields. This term chromosomics is encompassing the integration of the latest advances in cytogenetics, genome sequencing, epigenomics, and cell biology. Chromosomics approaches in future will lead to additional success in answering fundamental biological questions in hematologic malignancies.

Next-Generation Karyotyping

In early 2021 three studies used novel next-generation karyotyping which employed WGS as a potential replacement for conventional cytogenetics ( Chapter 3 ). These initial studies focused on patients with AML and myelodysplastic syndrome (MDS) and the genetic profiles and sequencing approaches were tested against the backbone of orthogonal results obtained at the same time using cytogenetics, FISH, and polymerase chain reaction (PCR) and/or NGS for detection of acquired somatic mutation. In a study reported by the Washington University group, in 235 patients who had undergone successful cytogenetic analysis, WGS provided rapid and accurate genomic profiling in patients with AML or MDS after modifications of sample preparation, sequencing, and analysis to detect mutations to be used for risk stratification using existing European Leukemia Network (ELN) guidelines. The WGS detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, next-generation karyotyping identified new previously unrecognized genomic events in 17% of patients. In 117 consecutive patients analyzed prospectively WGS provided results within a median of 5 days and additional genetic information was identified in almost 25%, which changed the ELN and Revised International Prognostic Scoring System (IPSS-R) genetic prognostic risk categories in 16% of patients. Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. WGS was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. These initial data are highly promising because they provided a greater diagnostic yield than conventional cytogenetic analysis and allowed for more efficient risk stratification on the basis of standard risk categories. Moreover, the speed by which the clinically relevant genomic profiles were obtained allowed reports to be generated in as little as 3 days.

Clonal Origin of Leukemia

The question of whether cell proliferation is monoclonal or polyclonal 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. 57.7 ). 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 chromosome 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.

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. 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 chromosome 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.

In Utero Mutations and Clonal Origin of Hematologic Malignancies

The observation that monozygotic twins share identical but non-constitutive and clone-specific fusion gene sequences (e.g., ETV6-RUNX1 ) in pediatric ALL provided the first unambiguous evidence (in 2003) that genetic lesions, generated by chromosomal translocation, arise in utero. These studies initiated by Mel Greaves and his colleagues contributed a wealth of knowledge about founder mutations, subclonal development, clonal origin, and the evolution of disease. The initiating lesion and premalignant clone is shared by the twins as a consequence of intraplacental vascular anastomoses and blood cell chimerism. The twin data were endorsed by backtracking of prenatal-initiating genetic lesions in the archived blood spots, or Guthrie cards, of patients with ALL. These data were interpreted to suggest that ETV6-RUNX1 was likely a critical initiating lesion for ETV6-RUNX1 -positive ALL. However, such fusions were detectable in cord blood from newborn infants at rates approximately 100-fold higher than the incidence of ALL, suggesting an obligatory requirement for additional mutations for leukemia development to occur. Over a period of 10 years these results were confirmed and the in utero origin of MLL , BCR-ABL1 , and RUNX1-RUNXT1 fusion rearrangements further documented, providing direct evidence for a prenatal origin of many childhood leukemias.

Results from genome sequencing of ETV6-RUNX1 fusion region suggests it arises as a consequence of nonhomologous end-joining in the pro-B-cell stage with possible self-renewal capacity to downstream B-cell precursors. Additional evidence was provided by screening for trisomies in stored cord blood and the data indicated that ∼6% of enriched CD34+/CD19+ B progenitor cells carry trisomies frequently seen in hyperdiploid childhood ALL. Using novel SNP as well as other technologies, it has become apparent that ALL has multiple genome copy number variations (CNVs), mostly deletions, and these CNVs are distinctive between a pair of twins, indicating a secondary, postnatal origin.

When genotyping sequencing combined with other molecular technologies of five pairs of monozygotic twins with concordant ETV6-RUNX1 -positive ALL, Greaves and his colleagues demonstrated that all recurrent CNVs (32 in total) were different within twin pairs providing strong evidence that they are probably secondary events and postnatal in origin in both twins as well as in non-twins with ALL. Another two twin pairs who shared a monochorionic placenta and had Ph-positive ALL, were also studied by Greaves and his colleagues. These studies provided confirmation of the previous observation that BCR-ABL1 is not sufficient to cause Ph-positive leukemia. Twin A presented with ALL at age 3.8 years and twin B presented at age 4.1 years. Both had an identical BCR-ABL1 fusion transcript and both received an allogeneic stem cell transplantation from the same matched sibling donor but 7 months later twin B died, whereas twin A remained in good health 8 years following the transplantation. SNP analysis revealed that twin A had a subclone with trisomies for chromosomes 4, 6, 9, 14, 17, and X, tetrasomy 21, and a gain of 22q11.1–q11.23 region, whereas twin B had deletions of EBF1 and IKZ1 . These results provide direct evidence that rearrangements additional to BCR-ABL1 are postnatal in origin, they represent subclonal evolution, and BCR-ABL1 by itself is not sufficient for development of Ph-positive ALL. Recently two groups have reported, using mathematical models, that some patients’ MPNs acquire the JAK2 mutation in utero.

These mutation-driven natural history studies provide evidence for sequential multistep pathogenesis of hematologic malignancies with sequential accumulation of genetic changes, which may be linear through clonal succession but that clonal evolution in most leukemias is complex and may represent a branching structure, as predicted by Darwin.

Chronic Myeloid Leukemia (see Chapter 69 )

Knowledge of the origins of CML has accumulated over the last 61 years and serves as a classic example of molecular medicine at its best ( Table 57.3 ). The Ph chromosome is the first example of a specific chromosomal abnormality associated with a malignant disease. ABL1 and BCR genes are the first oncogenes localized at the site of a chromosomal breakpoint in t(9;22)(q34;q11.2). The BCR-ABL1 fusion leads to a “hybrid” gene, resulting in the production of a dysregulated tyrosine kinase protein. Finally, imatinib mesylate, a specific tyrosine kinase inhibitor (TKI), was the first rationally designed targeted form of cancer therapy.

The Ph chromosome, named in honor of Philadelphia, the city of its discovery, was described for the first time in 1960. It represents a signature genomic rearrangement occurring in more than 95% of patients with CML. Approximately 3% of all pediatric leukemias are Ph-positive CML. The incidence in children increases with age and it is exceptionally rare in infants. The Ph chromosome results from a balanced translocation t(9;22)(q34;q11.2) ( Fig. 57.8A–B ). The Ph chromosome arises postzygotically, being found only in hematopoietic tissue. The finding of the Ph chromosome in myeloid cells, erythroid cells, eosinophils, monocytes/macrophages, basophils, and B lymphocytes, along with the absence of the Ph chromosome in cultured marrow fibroblasts, supports the concept that the Ph chromosome results from a specific rearrangement in a multipotent hematopoietic stem cell and that it is an acquired rather than an inherited abnormality. Of interest, the Ph chromosome is rarely identified in T cells. T lymphocytes are long-lived cells and may antedate the development of CML. These observations combined with studies exploiting G6PD heterozygosity provide further evidence for the concept that CML is a clonal disease arising in a stem cell capable of differentiation into all hematopoietic cell lineages.

In a review of 1129 Ph-positive patients, the 9;22 translocation was identified in 1036 (92%) cases. Karyotypic analysis of marrow cells in patients with CML is a time-consuming task. However, it demonstrates not only the presence of the Ph chromosome but also the presence of other chromosomal rearrangements (clonal evolution) of clinical significance.

The reciprocal nature of the Ph-positive translocation was confirmed when studies showed that it is the molecular consequence of the translocation of the ABL1 gene from chromosome 9, band region q34, and subsequent fusion to the breakpoint cluster region ( BCR ) gene on chromosome 22, band q11.2 ( Fig. 57.9 ). This creates a hybrid BCR-ABL1 gene that is transcribed into a chimeric BCR-ABL1 messenger RNA (mRNA) and translated into a specific chimeric protein.

Three major breakpoint locations along the BCR gene on chromosome 22 result in three chimeric proteins. They include P210 ^BCR-ABL1 , P190 ^BCR-ABL1 , and P230 ^BCR-ABL1 which are associated with three distinct types of leukemia. P210 ^BCR-ABL is found in the majority of patients with classic Ph-positive, BCR-ABL1 fusion-positive CML and approximately 30% of patients with Ph-positive ALL. Expression of P190 ^BCR-ABL1 is seen in 20% to 30% of adults and 80% of children with Ph-positive ALL. Expression of P230 ^BCR-ABL1 is associated with a rare indolent chronic neutrophilic leukemia variant and up to 1.6% of CML (approximately 50 patients in the worldwide literature have been described). The most common BCR-ABL1 isoform results from a fusion of BCR exon 13 or 14 (e13/e14) with ABL1 exon 2 (a2) producing a fusion gene referred to as e13a2 (b2a2) or e14a2(bra2) producing above-mentioned p210 protein. The coiled-coil domain of the BCR N terminus facilitates dimerization and constitutive autophosphorylation of the ABL1 tyrosine kinase domain resulting in subsequent phosphorylation of numerous substrates including the JAK2/STAT CRKL MAPK and P13K/AKT pathways.

Approximately 1% to 2% of Ph-positive patients with CML express both P210 ^BCR-ABL1 and P190 ^BCR-ABL1 and their response to TKI therapy is inferior to patients showing only P210 ^BCR-ABL1 . The BCR-ABL1 fusion is present in both standard and variant forms, in cases where chromosome 9 involvement is cytogenetically not detectable, and when a masked Ph is present. In the majority of patients, the fusion of ABL1 and BCR takes place on chromosome 22 ( Fig. 57.10A–B ). The BCR-ABL1 fusion transcript is present in neutrophils, monocytes, eosinophils, erythrocytes, B cells, rarely in T cells, and in CD34+ cells and is associated with increased proliferation of CD34+ myeloid progenitor cells but not of other more mature myeloid precursors.

These observations confirm the hypothesis that CML originates in a multipotent stem cell capable of differentiating to all hematopoietic cell lineages with the exception of T cells. These and other studies provide evidence for the existence of clonal BCR-ABL1 fusion-negative stage. The formation of BCR-ABL1 and the Ph chromosome occurs in an already abnormal and genetically unstable clone of pluripotent hematopoietic stem cells. Thus it is the preexisting genetic instability that predisposes to the formation of the Ph chromosome. Once Ph chromosome formation occurs, it confers a further selective growth advantage over normal cells, resulting in overwhelming BCR-ABL1 -positive, Ph-positive marrow cells at the time of diagnosis of CML.

In 5% to 8% of newly diagnosed patients with CML, the Ph chromosome is present as a 3- or 4-way complex derivative Ph chromosome, always involving chromosomes 9 and 22 as well as an additional one or two chromosomes. In our own laboratory we have observed over many years at least 25 different complex Ph translocations. Three-way translocations are more frequent than 4-way translocations. Some of the examples are the following translocations ( Fig. 57.11 ): t(2;9;22)(p13;q34;q11.2), (9;22;7)((q34;q11.2;p15), t(9;19;22)(q34;p12;q11.2), t(5;9;22) (q35;q34;q11.2), t(9;22;8)(q34;q11.2;p21), t(9;11;22)(q34;p15;p11.2), t(9;22;22) (q34;q11.2;p11.2), t(6;9;22;10)(q25,q34;q11.2;q24), t(7;9;22;22)(q22;q34;q11.2;q13.2), t(6;9;22;11)(q25,q34;q11.2;q13). Most of these complex structural rearrangements are unique but some of them such as t(2;9;22) are recurrent. Moreover, there are less than 1% of CML patients in chronic phase who acquire the t(9;22) clone abnormalities as well as t(8;21), inv(16), t(11q23), t(15;17), and t(3;3) that are associated with AML. In one recent retrospective study among 1382 Ph-positive patients 0.36% of patients had co-occurrence of these abnormalities and most of them progressed to accelerated or myeloid blast crisis.

In about 3% to 5% of patients with CML who are Ph-negative by cytogenetic studies, the clonal and stem cell origin of these hematologic malignancies can still be demonstrated, and molecular analysis reveals the BCR-ABL1 fusion in approximately 1% ( Fig. 57.12 ). These patients have chromosomal insertions resulting in BCR-ABL1 fusion which can occur in multiple ways. Approximately 70% of cases exhibit BCR-ABL1 fusion located on a chromosome 22, which was either a cryptic der(22), or “cryptic Ph” or non-cryptic der(22) or “masked Ph.” The remaining cases have BCR-ABL1 fusion located on a cryptic or non-cryptic der(9). Of the 119 such cases reported in literature 77 patients (64.7%) had an insertion-derived BCR-ABL1 fusion as a cryptic or a masked Ph. This type of BCR-ABL1 fusion does not influence OS of these CML patients. In the majority of Ph-negative patients, an ABL1 insertion from chromosome 9 to 22q11.2 results in a BCR-ABL1 fusion product without reciprocal translocation of sequences from chromosome 22 to chromosome 9. Sizes of the inserts can vary, which may be closely related to the morphological appearance of the chromosomes with the BCR-ABL1 or those involved chromosomes without the BCR-ABL1 . Intensive FISH mapping using many probes targeting ABL1 , BCR , and their flanking regions determined that a classic t(9;22) usually involves an exchange of approximately 10 MB of 9q (from the ABL1 to the 9q telomere) and ∼25 MB of 22q (from BCR to the 22q telomere).

Approximately 2% of patients truly are Ph-negative and BCR-ABL fusion-negative. These patients may not have CML but rather another MPN ( Chapter 71 ). The concept that the BCR-ABL1 fusion plays a central role in the pathogenesis of CML is strongly supported by two lines of evidence: (a) retroviral transduction experiments in which P210 ^BCR-ABL1 is expressed in murine marrow cells, resulting in an MPN resembling CML, and (b) the fact that imatinib, a TKI, selectively inhibits the BCR-ABL1 fusion protein in mice and specifically inhibits the growth of human Ph-positive cells in vitro and in vivo. Although considered necessary, BCR-ABL1 may not be the initial event or be sufficient to cause the malignant transformation resulting in CML (see earlier section on clonal origin).

PCR analysis can determine the exact breakpoints of DNA fusion products. Reverse transcriptase PCR (RT-PCR) and Northern blot analysis allow detection of BCR-ABL1 transcripts at the RNA level. Current FISH studies can detect BCR-ABL1 fusion at diagnosis using a dual-color BCR-ABL1 ES probe or BCR-ABL1 dual-fusion probe (see Fig. 57.12 , see also Fig. 57.9 ). A triple-color BCR-ABL1-ASS probe is used for detection of deletions on both chromosomes 9 and 22 ( Fig. 57.13 ). Approximately 12% to 15% of patients with CML have large deletions adjacent to the Ph chromosome translocation breakpoint on the derivative 9 chromosome. These deletions are heterogeneous and may involve both chromosomes 9 and 22 (majority of cases), only chromosome 9 (8% of patients with deletions), or only chromosome 22 (4% of cases with deletions). Moreover, deletion size is variable, ranging from 0.5 Mb to greater than 10 Mb. A more refined study applying SNP microarrays revealed three common deleted regions: (1) a 162-kb loss at 9q34, (2) a 138-kb deletion at 22q11.2, and (3) a 102-kb deletion at 22q11.2. It appears that the partial deletion of the ABL1-BCR fusion on derivative 9 chromosome occurs as a part of the same process as the formation of the BCR-ABL1 translocation. Before imatinib therapy, these deletions were associated with an adverse prognosis; however, in a study of 521 patients with CML, the cumulative incidence of complete cytogenetic responses and major molecular responses and the overall survival (OS) were comparable after 5 years of follow-up between CML patients with and without the del(9q).

At diagnosis, conventional cytogenetics remains the gold standard because the chromosome analysis will identify not only the t(9;22) but also other chromosomal abnormalities that may indicate accelerated or blast phase of the disease or clonal proliferation of Ph-negative cells.

Imatinib mesylate (Gleevec) has revolutionized therapy for CML and, for most patients, has transformed a deadly disease into a chronic disorder that is compatible with normal life. The standard method for monitoring a patient’s response to therapy is conventional cytogenetic analysis of cells obtained from a marrow aspirate. In the phase III international Randomized and STI-571 (IRIS) study, 89% of patients had Ph-negative marrow aspirates as determined by conventional cytogenetics after 5 years of treatment. However, conventional cytogenetics has limited sensitivity. The degree of MRD is determined to be an important prognostic factor for patients with CML on therapy. Patients with CML without a discernible Ph chromosome detected by conventional cytogenetics analysis may still harbor up to 10 ¹⁰ leukemic cells.

Interphase FISH does not depend on the cycling status of cells, and use of double-fusion probes has reduced false-positive results to approximately 1%. However, if peripheral blood cells rather than marrow aspirate cells are used to monitor MRD, the high percentage of BCR-ABL1 fusion-negative lymphoid cells may underrepresent the actual residual tumor load. In most direct comparison studies, interphase FISH of peripheral blood compared with conventional cytogenetics of marrow in patients who are treated with imatinib showed good correlations ( r = 0.91 to 0.97). Real-time quantitative PCR (RQ-PCR) is by far the most sensitive method for detecting MRD. It provides an accurate measure of the total leukemia cell mass and the degree to which BCR-ABL1 transcripts are reduced by therapy, and it correlates with progression-free survival.

The goal of therapy in CML is to achieve a molecular remission as measured by the reduction or elimination of BCR-ABL1 transcripts. In the IRIS study, at 5-year follow-up, complete cytogenetic response combined with major molecular response at 12 months was associated with a 97% progression-free survival rate. This compares with an 89% progression-free survival for those with complete cytogenetic response but without a major molecular response. Current international recommendations for optimal molecular monitoring of patients receiving imatinib treatment includes an RQ-PCR assay expressing the BCR-ABL1 transcript levels on an internationally agreed upon scale. The term major molecular response corresponds to ≤0.0% BCR-ABL1, whereas the designation complete molecular response should be used only for patients with undetectable BCR-ABL1 , where the limit of detection is confirmed to be at least a 4.5 log reduction from baseline levels. The two major obstacles to successful imatinib-based therapy for patients with Ph-positive, BCR-ABL1 fusion-positive CML are the persistence of BCR-ABL1 fusion-positive cells and relapse of the disease because of emergence of resistance to imatinib. Acquired resistance to imatinib treatment can be due to two events: amplification of BCR-ABL1 fusion product ( Fig. 57.14 ) and mutations in the ABL kinase domain. Currently, 100 different ABL1 kinase domain mutations have been described, although only about 15 are common and account for more than 85% of all mutations detected.

Patients with CML who cannot tolerate or are resistant to imatinib may benefit from the second and now third generation of TKIs ( Chapter 69 ) such as dasatinib, nilotinib, bosutinib, and ponatinib. These agents bind to the ABL kinase domain in a matter distinct from that of imatinib and thereby retain activity against nearly all imatinib-resistant mutations. Recent meta-analysis revealed ABL mutation rates with imatinib 9.7%, dasatinib 1.7%, nilotinib 3.3%. The most common specific mutations were T315I, E255k, and M315I. T315I mutations constitute 58% of dasatinib-related mutations and 13% of imatinib-related mutations. These mutations inhibit the binding of the TKIs, hindering the treatment of patients with CML. The introduction of ponatinib, a new formulation of TKI, has been shown to overcome resistance incurred by the T315I mutation (see Chapter 69 ).

Between 5% and 8% of patients undergoing treatment with imatinib will develop additional chromosomal abnormalities (ACAs) such as trisomy 8, monosomy 7, del(20q), and other anomalies in BCR-ABL1 fusion-negative cells. Imatinib may induce chromosomal abnormalities in BCR- ABL1 ⁻ cells. Alternatively, imatinib may uncover chromosomal abnormalities present before therapy after significant reduction of Ph-positive cells has occurred ( Fig. 57.15 ). Presence of +8 and other chromosomal anomalies in Ph-negative cells in patients treated with imatinib suggests that CML has a multistep pathogenesis and that clonal Ph-negative cells precede the development of the Ph-positive clone ( Fig. 57.16 ). This important observation about the pathogenesis of CML demonstrates the power of conventional cytogenetics, even in the era of molecular assays, and should be used at least annually while patients are undergoing TKI treatment. This hypothesis that clonal development occurs in Ph-negative cells before development of the Ph chromosome was recently confirmed using NGS by targeting 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities during TKI treatment. Mutations affecting DNMT3A , EZH2 , RUNX1 , TET2 , TP53 , U2AF1 , and ZRSR2 were detected in 43% of these patients. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. Moreover, somatic mutations in addition to BCR-ABL1 affecting ASXL1 , DNMT3A , RUNX1 , and TET2 genes are found in 33% of patients. When individual hematopoietic colonies were analyzed from patients with CML at diagnosis, most mutations were present in the Ph-positive cells. By contrast, NGS of subsequent samples during TKI treatment revealed that one DNMT3A mutation occurred in Ph-negative cells that was also present in Ph-positive cells at diagnosis, strongly implying that DNMT3A preceded the appearance of the BCR-ABL1 rearrangement which provides support for the initial hypothesis that BCR-ABL1 and the formation of the Ph chromosome were not the initiating events in the pathogenesis of CML. Another example of clonal evolution in MPNs evolving over time is the co-occurrence of the JAK2V617F mutation and BCR-ABL1 . These rare patients usually have a long (10 to 20 years) history of JAK2V617F+ polycythemia vera (PV) before BCR-ABL1 and the Ph chromosome is acquired. Single cell genotyping revealed that both JAK2V617F and BCR-ABL1 can occur concurrently in hematopoietic stem cells and that JAK2V617F+ occurs before the acquisition of BCR-ABL1 . The contribution of BCR-ABL1 to disease progression appears to be greater than that of JAK2V617F , since these patients display a clinical phenotype that is consistent with CML rather than PV. Recent studies have demonstrated the presence of somatic mutations in TET2 , DMNT3A , and ASXL1 in both diagnostic and/or follow up remission CML specimens implying evolution within the Ph-negative clone and indicating that these mutations are early events which preceded formation of the BCR-ABL1 gene. The mechanism of transformation to advanced-phase CML is complex and varied and remains poorly understood. In myeloid blast crisis (70%) of CML patients show karyotypic evolution, that is, new chromosomal abnormalities in very distinct patterns in addition to the Ph chromosome. The most common abnormalities include gain of chromosome 8 (33%) or +19 (12%), gain of a second Ph chromosome (30%), i(17q)(20%), alone or in combination, to produce modal chromosome numbers of 47 to 50 ( Figs. 57.17 and 57.18 ). In males, LOY is frequently observed. Isochromosome 17q occurs almost exclusively in myeloid blast crisis. Other less frequently observed abnormalities include monosomy of chromosomes 7 and 17, and trisomy of chromosomes 17 and 21. In addition to these common karyotypic evolutions, additional chromosomal aberrations specific for AML—for example, t(8;21) ( Fig. 57.19 ), inv(16), t(3;21)(q26;q22), and most frequently, inv(3q)/t(3;3), resulting in the overexpression of MECOM (EVI1) —have been observed. Simultaneous presence of t(9;22) and inv(3) at diagnosis or following TKI therapy appears to be associated with a lack of response to treatment and a clinical course is similar to that of cases with inv(3) without any other chromosomal abnormality. In patients treated with TKI, these ACAs have been classified into two groups based on their impact on survival. ACAs in the Good prognostic group include trisomy 8, LOY, and an extra copy of the Ph chromosome. The second group includes chromosomal abnormalities that are associated with a relatively poor prognosis including i(17q), −7/del(7q), and 3q26.2 rearrangements. The concurrent presence of two or more ACAs was associated with an inferior survival. A prospective study of 1551 imatinib-treated chronic phase patients (German CML-study IV) defined more precisely a high-risk and a low-risk group based on the presence of ACAs. High-risk chromosome abnormalities and their impact on survival included +9,+Ph, i(17q),+17,+19, 3q26.2, 11q23, −7/del(7q), and a complex karyotype, while the low-risk group included all other karyotypic changes. High-risk abnormalities were observed in 6% of patient during chronic phase but in 61% of patients who progressed to blast crisis while low-risk chromosome abnormalities were observed in 21% during CP and in 2.8% of patients who progressed. Most importantly, this study demonstrated that the combination of high-risk cytogenetic abnormalities and a low-level blast cell count (1% to 5%) was associated with disease progression and death from CML. Therefore, additional high-risk chromosomal abnormalities are an early marker of CML progression even when present in patients with low numbers of blast cells.

Complete cytogenetic remission for patients in accelerated phase or blast crisis CML treated with imatinib is rarely accompanied by a normal karyotype; however, 6% to 17% of these patients may have some cytogenetic response (see box on Genetic Testing for Chronic Myeloid Leukemia ).

High-resolution cytogenomic studies have demonstrated that in contrast to the chronic phase CML (which is characterized only by genomic imbalances in or around BCR and ABL1 , specifically deletions), during the blast phase, many additional genomic imbalances occur. Using aCGH, 44 patients in chronic phase CML as well as 11 in myeloid and in lymphoid blast crisis were investigated, and a spectrum of recurrent genomic imbalances was associated with disease progression, including losses at 1p36, 5q21, 9p21, and 9q34 and gains at 1q, 8q24, 9q34, 16p, and 22q11. The most common mutations in myeloid blast phase involve tumor suppressor gene TP53 (about 25% of cases) and the RUNX1 in about 40% of cases. Moreover, the MYC oncogene has been recently linked to BCR-ABL1 activity and CML progression to myeloid blast crisis. Recently, mutations in genes frequently found in Ph-negative MPNs such as CBL , TET2 , ASXL11 , and IDH1/2 were also identified in patients in accelerated/blast phase. Additionally, deletions of RB1 , gain of function mutations in GATA-2 and RAS are just some of the spectrum of cytogenomic changes characterizing progression of CML.

Patients with CML may present initially in lymphoid blast crisis mimicking Ph-positive B-cell ALL. Although both the CML and Ph-positive ALL (see later) are driven by the constitutively active BCR-ABL1 tyrosine kinase, their prognoses differ significantly and it is important to differentiate these two entities. Cytogenomic analysis of 78 CML patients in lymphoid blast crisis demonstrated a unique signature of genomic deletions within the Ig heavy-chain and TCR genes, indicating that their presence is essential for the development of a malignant clone with a lymphoid phenotype. Moreover, 50% of patients in lymphoid blast crisis had mutations in cyclin-dependent kinase inhibitor CDKN2A , 25% to 35% in RUNX1 , and ∼15% to 25% have mutation in the BCOR gene, which interacts with histone deacetylates, a known apoptosis regulator. One of the most important differences between lymphoid and myeloid blast crisis is the exclusive interaction with STAT proteins. STAT1 is activated mainly by p190, while STAT3 and STAT5 are preferentially activated by the P210 isoform.

The precise causes and mechanisms responsible for CML progression are still elusive and current thinking is that progression of CML is the result of increased and complex genomic instability combined with heterogeneity of numerous coexisting mutations and signaling disruptions causing defective or an insufficient ability to repair DNA.

Ph-Negative Chronic Myeloproliferative Neoplasm

Ph-negative MPNs are disorders arising in a single clone of multipotent precursor cells in which one or all myeloid lineages are abnormally amplified. Classic and more frequently encountered Ph-negative MPNs include PV, primary myelofibrosis, and essential thrombocytopenia (ET). Their cytogenetic and genomic profiles are described in Chapters 70–73 . However, included in this chapter are Fig. 57.20A and B and 57.21 in order to illustrate the most frequent abnormalities associated with the Ph-negative MPN. Different 9p chromosomal abnormalities result in JAK2 numerical gain (see Fig. 57.20 ) while nine examples of 1q gain, associated with disease progression, are shown in Fig. 57.21 .

Less frequently encountered similar disorders include chronic neutrophilic leukemia ( Chapter 72 ), hypereosinophilic syndrome/chronic eosinophilic leukemia ( Chapter 74 ), systemic mastocytosis ( Chapter 75 ), atypical CML, and unclassifiable MPNs ( Chapter 72, Chapter 73, Chapter 74, Chapter 75 ). Rare recurrent balanced abnormalities in atypical Ph-negative MPNs can involve the PDGFRB gene localized at 5q33, FGFR1 gene on 8p11, and PDGFRA gene on 4q12. The best described are t(5;12)(q33;p13), resulting in ETV6-PDGFRB fusion protein, and t(8;13)(p11;q12), resulting in the ZNF198-FGFR1 fusion gene. The disorder known as 8p11 myeloproliferative syndrome has been classified by WHO as belonging to the group of myeloid/lymphoid neoplasms associated with eosinophilia and gene rearrangements. Some of these diseases are related to fusion genes between FGFR1 located on 8p11 and various translocations fusing at least 14 different 5′ partner genes to the 3′ part of the FGFR1 gene that encodes tyrosine kinase domain ( Table 57.4 ). They include TPR (1q25), LRRFIP1 (2q37), SQSTM1 (5q35) FGFR10P (6q27), TRIM24 (7q34), CUX1 (7q22), PLAG1 (8q12), CEP110 (9q33), NUP98 (11p15 ), FGFR1OP2 (12p11), CPSF6 (12q15), ZMYM2 (13q12), MYO18A (17q23), HERVK (19q13), and BCR (22q11) ( Fig. 57.22 ). The most frequent translocation is t(8;13), and cases with the submicroscopic deletion of the 5′ part of the FGFR1 gene have also been reported, similar to the formation of t(9;22) with deleted sequences from der(9q) and/or 22q identified in CML. The presence of 8p11 abnormalities in both myeloid and lymphoid cells suggests its origin from a common stem cell.

In patients with chronic eosinophilic leukemia/hypereosinophilic syndrome ( Chapter 74 ) the most frequent recurrent abnormality is the cryptic deletion on 4q12 as a result of FIP1L1-PDGFRA fusion gene reported to occur in from 3% to 56% of cases. The disparity in frequencies reflects differing levels of stringency criteria in the diagnosis of disorders with hypereosinophilia as well as different technologies used for detection of FIP1L1-PDGFRA fusion. An investigation of 376 patients with persistent unexplained hypereosinophilia revealed an 11% incidence of the FIP1L1-PDGFRA fusion gene detected using highly sensitive RQ-PCR. Patients with FIP1L1-PDGFRA fusion are characterized by a male predominance, marrow fibrosis, increased number of mast cells, elevated serum tryptase levels, and a favorable response to low doses of imatinib. Most of these patients have a normal karyotype because a cryptic deletion of CHIC2 locus on 4q12 which is only 800 kb in size. This abnormality is detectable using a more sensitive FISH technology and RQ-PCR in the majority of cases. Moreover, the commercially available tricolor FISH probe will detect not only deletion 4q12 as a result of FIP1L1-PDGFRA but also a rare BCR-PDGFRA fusion resulting from the t(4;22)(q12;q11.2) rearrangement ( Fig. 57.23 ). Serial monitoring with RQ-PCR demonstrates exquisite sensitivity of FIP1L1-PDGFRA -positive patients to low-dose imatinib treatment ( Chapter 74 ). The FIP1L1-PDGFRA has also been detected in histopathologically defined cases of systemic mastocytosis with associated eosinophilia, and in cases of AML and T-cell lymphoblastic lymphoma associated with eosinophilia. In an Italian prospective cohort of 27 patients with FIP1L1-PDGFRA who were treated with imatinib, a complete hematologic response was achieved within 1 month and all patients became PCR-negative for FIP1L1-PDGFRA after a median of 3 months (range 1 to 10 months). In contrast to CML, very few cases of acquired imatinib resistance have been reported. Those rare reported cases had a mutation in T674I within the ATP-binding domain of PDGFRA, analogous to the T315I mutation in CML.

Although translocations involving PDGFRB at 5q32 region have been identified with 22 different fusion partners, they are indeed very rare. Any patient with a 5q31–q33 chromosomal abnormality with or without eosinophilia should be investigated for PDGFRB rearrangements by FISH testing ( Fig. 57.24 ).

Once the PDGFRA, PDGFRB , and FGFR1 rearrangements are excluded, the only other recurrent secondary abnormalities include trisomy 8 and trisomy 21 (see box on Genetic Testing for Ph-Negative Myeloproliferative Neoplasms ).

In the WHO 2016 classification, mastocytosis is no longer listed as a Ph-negative MPN ( Chapter 56, Chapter 75 ) but rather as a distinct category, “Mastocytosis.” Mastocytosis includes a group of rare and heterogeneous diseases characterized by accumulation of clonal mast cells in one or multiple organs. Over 80% of patients with systemic mastocytosis ( Chapter 75 ) are characterized by KIT mutations, specifically in the activation loop of KIT , KIT D816V. KIT is encoded by a 21-exon-containing gene located on the long arms of chromosome 4, 4q12. Other rare KIT mutations have been described in adult and pediatric patients. The knowledge of the type and structure of KIT mutation is important because the A-loop mutations D816V/H/Y/N disrupt the structure of the receptor, leading permanently to an active conformation and resistance to imatinib. In patients with advanced systemic mastocytosis additional mutations have been observed including TET 2 (up to 39%), SRSF2 (up to 36%), ASXL1 (up to 21%), and RUNX1 (23%). At diagnosis of mastocytosis patients testing for KIT D816V in peripheral blood leukocytes, using highly sensitive and specific PCR-based assays, is necessary to determine the KIT allelic burden rapidly, which is now used as a gold standard for assessing disease burden.