The Cytogenetics of Hematologic Neoplasms

Fig. 15.1
G-banded karyogram of a normal bone marrow cell

Table 15.1
Glossary of cytogenetics terminology used in this chapter

Acentric fragment

A chromosome fragment lacking a centromere and therefore incapable of attaching to the spindle. Acentric chromosomes are distributed randomly among daughter cells


Deviation of the chromosome number that is characteristic for a particular species caused by either gain or loss of one or more chromosomes


Any chromosome other than the sex chromosomes


Alternating intrachromosomal light and dark segments along the length of chromosomes


Specific band on a chromosome containing a break in the DNA as the result of a chromosome rearrangement


An area of chromosomal constriction that holds the two chromatids together and is needed for spindle site attachment. Based on the position of the centromere, chromosomes are classified as metacentric (middle position), submetacentric (above the middle), and acrocentric (extremely small short arm consisting of satellites and stalks)


Arrangement of nuclear genetic material into formations containing a centromere and two chromosome arms. The normal chromosome number in human somatic cells is 46, whereas in germ cells it is 23

Chromosome rearrangement

Structural aberration in which chromosomes are broken and rejoined. These rearrangements can occur on a single chromosome or involve multiple chromosomes

Clonal evolution

A stepwise evolution characterized by the acquisition of new cytogenetic abnormalities


The examination of chromosomes


Loss of a chromosome segment. Deletions can either be terminal or interstitial


A chromosome containing two centromeres


Normal chromosome complement (two copies of each autosome and two sex chromosomes) in somatic cells

Double minute

Cytogenetic visualization of gene amplification. So called because of their appearance as two adjacent dots. Each double minute is thought to contain hundreds of copies of a particular oncogene


Two copies of the same segment present on a single chromosome


Half (i.e., 23 chromosomes) of the normal human chromosome complement in somatic cells. This is the number of chromosomes present in normal germ cells

Homogeneously staining region

Cytogenetic visualization of gene amplification. Multiple copies of a particular oncogene are inserted into one of more chromosome region giving the appearance of a uniform staining

Hybrid gene

Fusion of two different genes as a result of a structural chromosomal rearrangement. A hybrid gene leads to a hybrid protein with abnormal function


Gain of one or more chromosomes


Loss of one or more chromosomes


Diagrammatic representation of a partial or complete karyogram


Balanced or unbalanced relocation of chromosomal material into a different or the same chromosome


Structural rearrangement affecting a single chromosome. This is generated by a 180° rotation of a segment included between 2 breaks along a single chromosome. Inversions can be paracentric (breaks involving a single arm) or pericentric (breaks involving both arms)


Suggested guidelines of An International System of Human Cytogenetic Nomenclature used for the description of karyotypes


Structural rearrangement affecting a single chromosome generated by the misdivision of the centromere in transverse plane resulting in loss of one arm and duplication of the other


Arrangement of metaphase chromosomes according to size, position of centromere, and banding patterns


Description of the chromosome complement according to ISCN guidelines


Location of a particular gene on a chromosome

Marker chromosome

Chromosome whose origin cannot be identified using standard banding methods


Arrangement of chromosomes in one plane at the equator of the cell. This phase of mitosis is characterized by the disappearance of the nuclear membrane and appearance of the spindle with subsequent attachment of the centromeres to the spindle


The absence of one member of a homologous pair of chromosomes


Gene that promotes cell growth and development. One abnormal allele is sufficient to cause uncontrolled growth and lead to tumor formation


A cell containing a multiple of the haploid chromosome complement


Approximate diploid number of chromosomes, often accompanied by structural rearrangements

Recurrent abnormality

Structural rearrangement or numerical abnormality detected in multiple patients with the same or similar disease

Ring chromosome

A circular formation of a chromosome originating from two breaks on opposite arms and reunion of the broken ends

Sex chromosomes

The X and the Y chromosomes. With some exceptions, XX is observed in females and XY in males


Chromosome abnormality resulting from a break in two or more chromosomes and exchange of the material distal to the breaks. In a balanced translocation, there is exchange but no loss of DNA, whereas in an unbalanced translocation there is gain or loss of DNA. With unbalanced translocations, abnormal chromosomes are referred to as derivatives if the exchanged material is known. The term add is used if the origin of the exchanged material cannot be identified


Three copies of a chromosome

Tumor suppressor gene

Locus that inhibits tumor growth when at least one allele is functional.

Loss of both alleles is associated with tumor growth

Importance of Conventional Cytogenetics in the Diagnosis and Prognosis of Hematologic Neoplasms

There is no question that the development of sophisticated techniques such as fluorescence in situ hybridization (FISH), multicolor karyotyping (M-FISH, SKY), and, to some extent, array comparative genomic hybridization (array CGH) has enhanced the knowledge of chromosome abnormalities in hematologic neoplasms [1523] (see also Chaps. 17 and Chaps. 18). These techniques have immensely contributed to the discovery of significant cryptic rearrangements as well as to the detection of such rearrangements in nondividing cells of various tissue preparations. Their invention was seen as a potential competitor to conventional cytogenetics, due to their higher resolution. Nevertheless, several years after the introduction of these sophisticated technologies, conventional cytogenetic analysis is still the best method for the diagnosis of most hematologic neoplasms since it has the advantage of an overall examination of all chromosomes, compared to the more focused detection of abnormalities with the other molecular genetic methods. Undisputed, in fact, is the ability of conventional cytogenetics to identify related and distinct clonal populations, which is challenging for FISH and practically impossible for array CGH [24, 25]. Furthermore, the presence of abnormalities acquired during clonal evolution, an important indicator of disease progression, might be missed during a targeted FISH analysis [2629].

Chromosome Abnormalities in Hematologic Neoplasms

Cytogenetics began in 1956, when Tijo and Levan, and soon after them Ford and Hamerton declared that normal human cells contained 46 chromosomes and not 48, as previously believed (see Chap. 1) [30, 31]. From that point on, experimental work on cell cultures and banding was geared to the improvement of chromosome spreading and morphology and was presented in subsequent publications [4, 32]. It was the detection of the Philadelphia chromosome by Nowell and Hungerford, however, that definitively established that chromosome abnormalities in leukemia are acquired and as such they are present exclusively in the neoplastic cells [4]. But it was not until the middle 1970s that reports of cytogenetic abnormalities in cancer started to increasingly populate the scientific literature [3335]. Today, a complete list of these abnormalities can be found in Mitelman Database of Chromosome Aberrations in Cancer [36]. It is immediately evident from consulting Mitelman’s database that the most common rearrangements in hematologic neoplasms are balanced translocations [3739]. In the majority of cases, translocations represent the sole abnormality, whereas in other cases, they are identified during disease progression [4044]. The significance of a primary translocation versus a later-appearing abnormality differs, the latter usually suggestive of a more aggressive clinical course. Similarly, the significance of the same translocation in de novo and treatment-related hematologic neoplasms differs, with the latter, again, carrying a worse prognostic outcome and, in some cases, a greater resistance to therapy [45]. Balanced translocations are often the sole abnormality in the majority of acute and chronic myeloid leukemias and in a large number of acute and mature lymphoid neoplasms [46, 47]. It is interesting to note that the product of a translocation in leukemia is almost always a hybrid protein with abnormal function, whereas in lymphoma no hybrid protein is produced [4850]. In lymphoma, the relocation of an oncogene to a site under the control of an immunoglobulin promoter often leads to overproduction of a protein with oncogenic activity [5153]. Translocations appear to be less frequent in myelodysplastic syndromes and classical myeloproliferative neoplasms where partial or full unbalances, leading to loss of tumor suppressor genes and/or gain of oncogenes, dominate [5460]. Apart from balanced translocations, practically every abnormality known today has been observed in hematologic neoplasms, including ring chromosomes, double minutes (dmin), and homogeneously staining regions (hsr), which for some time were considered to be present exclusively in solid tumors [6166]. The specificity and recurrence of chromosome abnormalities in hematologic neoplasms have gained significance to the point that the latest version of the World Health Organization (WHO) guidelines focuses intensively on the genetic and cytogenetic features of hematologic neoplasms as predictors of diagnostic and prognostic outcome [67].

Myeloid Neoplasms

The classification of myeloid neoplasms has recently been modified [67]. This reclassification more than ever before takes into account the genetic and cytogenetic changes associated with these neoplasms. Consequently, neoplasms with similar morphologic and genetic features have been grouped together. The myeloid neoplasms include the myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), MDS/MPN, and acute myeloid leukemias. These are described in more detail in the following sections.

Myelodysplastic Syndromes

The term myelodysplastic syndrome (MDS) refers to a fairly heterogeneous group of hematopoietic stem cell neoplasms characterized by a series of similar features such as dysplastic cellular morphology, defect in cellular maturation, and increased risk of transformation into acute myeloid leukemia (AML) via a multistep process [68, 69]. MDS is rare in children as it makes up approximately 5% of the pediatric hematologic neoplasms. MDS occurs mainly in adults with a median age of 70 years, and although there is a risk for developing AML, about 50% of deaths occur as a result of unrelated causes, such as bleeding or infection [70].

There are two main types of MDS: primary or de novo MDS, and secondary or therapy-related MDS. Although secondary MDS occurs as a result of treatment with radiation and/or alkylating agents or treatment with DNA topoisomerase inhibitors for an unrelated malignancy, the initial insults leading to the development of primary MDS are still being debated. Some of the possible triggers include exposure to radiation, tobacco, and benzene.

Classification of MDS

Cytogenetic studies, which are routinely performed in patients with these neoplasms, are useful since chromosome abnormalities provide both diagnostic and prognostic information [57, 58, 70]. Table 15.2 describes the subdivision of MDS neoplasms according to the 2008 World Health Organization classification [67]. Chromosome abnormalities have been observed in approximately 50% of patients with de novo MDS and in as many as 90% of patients with therapy-related MDS. There appears to be a correlation between the frequency of chromosomal abnormalities and the severity of disease, and this is evident in this Table [57, 69]. About 25% of patients with low-grade MDS, such as refractory anemia and refractory anemia with ring sideroblasts, have an abnormal karyotype, compared with 50–70% of patients with refractory anemia with excess blasts (RAEB-1 and RAEB-2). The karyotypes observed in MDS are variable as they present with single or complex chromosome rearrangements [5658].

Table 15.2
Subdivision of MDS neoplasms according to the 2008 WHO classification and percent of chromosome abnormalities in each category


Marrow blasts (%)

Cytogenetics (%)



















MDS with isolated del(5q)



Childhood MDS






Abbreviations: RCUD refractory cytopenia with unilineage dysplasia, RARS refractory anemia with ring sideroblasts, RCMD refractory cytopenia with multilineage dysplasia, RAEB-1 refractory anemia with excess blasts-1, RAEB-2 refractory anemia with excess blasts-2, MDS-U myelodysplastic syndrome—unclassifiable, t-MDS therapy-related MDS

Chromosome Abnormalities in MDS

The most frequent chromosome abnormalities are complete or partial loss of chromosomes 5 and/or 7, deletions on the long arm of chromosome 20, and gain of chromosome 8 [36, 37, 7175] (see Table 15.3 and Fig. 15.2). In general, aggressive neoplasms are characterized by more complex karyotypes than those seen in low-grade MDS. Furthermore, as a general rule, dosage aberrations appear to be more represented in primary MDS, whereas balanced translocations are encountered more frequently in secondary MDS [56] (see Table 15.3). Among therapy-related MDSs, complex karyotypes with loss/deletion of chromosomes 5 and/or 7 together with deletions of 6p, 12p, and/or 16q are typical of alkylating agent-induced MDS, whereas balanced translocations involving 11q23 (MLL) and 21q22.3 (RUNX1) are associated with preceding therapy with DNA topoisomerase II inhibitors [7679].

Table 15.3
Recurrent chromosome abnormalities in primary MDS and t-MDS


Primary MDS


Fig. 15.2









3q21.3 rearrangements


3q26.2 rearrangements






























11p15.4 rearrangements


11q23 rearrangements


12p13 rearrangements








































+ indicates the presence of an abnormality and ++ indicates increased frequency


Fig. 15.2
Partial karyograms of recurrent abnormalities in MDS (Refer to Table 15.3 for additional information on the various rearrangements illustrated in this figure)

MDS with Deletion of 5q

The significance of del(5q) in MDS has to take into account not only the presence of this abnormality but also the associated morphologic picture [72, 75, 80]. The size of the deleted portion of the long arm of chromosome 5 is highly variable. The critical deleted region is approximately 1.5 Mb in size and is located at 5q31.2, where the EGR1 gene is located [75].

del(5q) can be associated with the so-called 5q− syndrome. In this hematologic syndrome, patients present with refractory macrocytic anemia and demonstrate hypolobulated micromegakaryocytic hyperplasia in the marrow [73, 80]. A female predominance has been noted (sex ratio: 1M/3F). The clinical course is said to be relatively indolent, with a very low-risk of developing acute leukemia. In the International Prognostic Scoring System (IPSS), del(5q) MDS patients are placed in the most favorable prognostic category [81, 82]. About 15% of patients do not fit into this category but still have a del(5q) as the sole abnormality [83, 84]. These cases do not appear to have the same favorable prognostic outcome, demonstrating the importance of the specific deletion for the prognosis and response to therapy [58, 85]. Similarly, del(5q) together with other abnormalities is no longer associated with the most favorable prognostic outcome that is typically seen in patients with 5q− syndrome. Deletion 5q and/or complete loss of chromosome 5 in the context of a complex karyotype is frequently seen in high-grade as well as therapy-related MDS [63, 65]. Here, deletions of 5q might be derived from unbalanced translocations with a variety of chromosome regions. The most common of these is a dic(5;17)(q11.2;p11.2), a result of which is loss of TP53 at 17p13.1, a marker of poor prognostic outcome in numerous neoplasms [86].

MDS with Deletion of 7q or Monosomy 7

Deletion of 7q/monosomy 7 has been viewed as a marker of poor prognostic outcome [56, 57, 81]. However, the 2007 prognostic score criteria places patients with this rearrangement in an intermediate risk [82]. As a sole abnormality, del(7q) occurs in approximately 1% of cases [87]. Three regions are most frequently deleted: 7q22, 7q31.1, and 7q31.3 [88, 89]. Some studies indicate that retention of band 7q31 may be found in patients with longer survival, suggesting that 7q31 might be the location of a tumor suppression gene [89, 90]. More often, del(7q) or −7 occurs as part of a complex karyotype (approximately 5–10% of cases), characterized by recurrent abnormalities that include one or more of the following: rearrangements of chromosome 3, −5/del(5q), del(6p), +8, +9, del(9q), del(11q), del(12p), del(17p), +19, del(20q), +21 [56]. Monosomy 7 as a sole abnormality is seen in pediatric patients with all MDS subtypes as well as in juvenile myelomonocytic leukemia (JMML) [91]. Loss of chromosome 7 is also seen in siblings with the so-called −7 syndrome and a predisposition to develop juvenile MDS [92, 93]. Literature shows that the lost chromosome 7 can come from either parent, suggesting that some other genetic defect that predisposes these children to lose one chromosome 7 is at the origin of this phenomenon [94, 95].

MDS with Trisomy 8

Gain of one copy of chromosome 8 is recurrent in all myeloid neoplasms. In MDS, it is found in over 10% of patients [9698]. According to both the old and new prognostic scoring systems for MDS, trisomy 8 is associated an intermediate risk when detected as the sole abnormality [97]. The presence of additional abnormalities generally worsens the prognostic outcome [96]. Trisomy 8 is often present as an additional abnormality, particularly in addition to del(5q). In about 2% of cases, four copies of chromosome 8 (tetrasomy 8) might be seen. These patients are given a high prognostic risk [99].

MDS with Other Chromosome Abnormalities

Rearrangements of chromosome 3, specifically bands 3q21.3 and/or 3q26.2, occur in about 5% of cases [100]. They have been observed in de novo AML, and in accelerated phase or blast crisis CML [101, 102]. The most common rearrangements include inv(3)(q21.3q26.2), t(3;3)(q21.3;q26.2), and del(3)(q21.3q26.2) [103]. Generally, these patients present with trilinear dysplasia in their bone marrow with dysmegakaryopoiesis. These rearrangements are associated with an adverse prognostic risk in both MDS and AML. This adverse prognosis probably correlates to the highly increased MECOM (EVI1, at 3q26.2) expression, detectable in the vast majority of these patients [104, 105]. Some patients with the 3q21.3q26.2 rearrangement do not have detectable MECOM expression, suggesting that the poor prognosis in these patients may be independent of such expression [106].

Another recurrent abnormality in MDS is del(17p). This deletion, which often is observed in the context of a complex karyotype, can be the result of various rearrangements, including simple deletions, unbalanced translocations, formation of an isochromosome, and monosomy 17 [86, 107]. Deletion of 17p is recurrent in myeloid disorders, mainly refractory anemia with excess of blasts (RAEB-1 and RAEB-2) and AML. About 30% of AML and MDS cases with 17p deletion are therapy related [108]. Deletion of 17p has been found to correlate with a particular form of morphological dysgranulopoiesis, sometimes associated with TP53 mutation [109].

The clinical significance of sex chromosome loss in the bone marrow of patients with hematologic neoplasms is still questionable [110, 111]. Loss of the Y chromosome is observed in approximately 10% of MDS cases, but since it is seen also in males of increasing age without evidence of a hematologic neoplasm, it is generally interpreted to represent an age-related phenomenon of no clinical significance [112]. However, it is interesting to note that some elderly males with MDS and loss of the Y chromosome show the Y chromosome in their marrow cells when they achieve complete hematologic remission. Loss of an X chromosome in the bone marrow of female patients is less ­frequent than loss of the Y chromosome in males, and tends to be viewed as being associated with a hematologic neoplasm rather than as an age-related phenomenon [113].

An interesting association is loss of the Y chromosome together with gain of chromosome 15, which is also characteristically seen in males with increasing age [114] (Fig. 15.3). The significance of trisomy 15 with or without the loss of the Y chromosome is not fully understood. In some cases, particularly when only a few abnormal metaphase cells are present, this finding is thought to be a transient phenomenon by some but not all authors [115117].


Fig. 15.3
Karyogram showing the simultaneous gain of chromosome 15 and loss of the Y chromosome (arrows). The significance of trisomy 15, particularly when present in few cells, is not clear

Apparently balanced translocations have been reported in MDS, but they appear to be less common than the unbalanced rearrangements. Chromosomes 1, 2, 3, 5, 6, 7, 13, 15, 17, 18, 19, and 20 appear to be more frequently involved [118]. Table 15.3 shows some of the most well-characterized translocations. From this Table, it is apparent that balanced translocations have been found in both de novo and therapy-related MDS.

Due to the variety of chromosome abnormalities reported in MDS, it is understandable that, at present, the best genetic test at diagnosis is conventional cytogenetics [119]. FISH is unquestionably useful when a limited number or no metaphase cells are available or as a follow-up tool for a patient with a known cytogenetic abnormality, but adds little to a normal conventional chromosome study based on the analysis of 20 metaphase cells [120].

Prognostic Significance of Chromosome Abnormalities in MDS

The 1997 International Prognostic Scoring System (IPSS; Table 15.4), which was constructed with data gathered from patients with de novo MDS, was revised in 2007 to also include patients who received established treatments for MDS [81, 82]. This new prognostic scoring system named IPSS-IMRAW (International MDS Risk Analysis Workshop) is a combined effort by European and American institutions and lists 22 groups of chromosome abnormalities compared with only 7 listed in the 1997 IPSS (Table 15.5). These guidelines are still a work in progress, and experts from around the world are intensively working on a more updated and satisfactory version.

Table 15.4
International Prognostic Scoring System of common abnormalities in MDS [81]



Median survival (mo.)

Normal, isolated  −  Y, isolated del(5q), isolated del(20q)



Complex with ≥3 abnormalities, −7/del(7q)



Other abnormalities



Table 15.5
Revised prognostic scoring system of common chromosome abnormalities in MDS based on the combined German-Austrian, Spanish MDS Registry, and IMRAW cohorts (IPSS-IMRAW) [82]



Median survival (mo.)

Normal, −Y, isolated del(5q), del(11q),del(12p), del(20q), t(11;V)(q23;V), +21, any 2 abnormalities including del(5q)



+1q, t(3q21.3;V), t(3q26.2;V),+8, t(7q;V), +19, −21, any other single abnormality, any 2 abnormalities not including 5q or 7q



−X, −7 or del(7q), any 2 abnormalities with −7 or del(7q), complex with 3 abnormalities



Complex with >3 abnormalities



V variable translocation partners

Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by proliferation of one or more myeloid cellular elements in the marrow and mostly affect adult individuals [121]. These neoplasms are known by different names, depending on the lineage affected. The classic MPNs include chronic myelogenous leukemia (CML), polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET) [122]. Other hematologic disorders included in the MPN category are chronic eosinophilic leukemia (CEL), systemic mastocytosis, chronic neutrophilic leukemia (CNL), and the unclassifiable MPNs [67, 123, 124]. Except for CML, which is characterized by the presence of the t(9;22)(q34;q11.2)—the Philadelphia (Ph) chromosome translocation—the classic MPN exhibits similar cytogenetic abnormalities, such as gain of 1q, +8, +9, del(13q), and/or del(20q) [59, 71, 125, 126]. Two or more of these abnormalities might be present in the same karyotype (Table 15.6).

Table 15.6
Classification of myeloproliferative neoplasms according to the WHO, including the most common chromosome abnormalities associated with them


Frequent abnormalities

Abnormalities during progression



+8, i(17q), +der(22)t(9;22)


+8, +9, del(20q)

−5/del(5q), −7/del(7q), del(17p)


+8, 13q−, del(20q)

+1q, −5/del(5q), −7/del(7q), del(17p)


+1q, +8, +9, del(20q)

+1q, −5/del(5q), −7/del(7q)


4q12 rearrangements (KIT mutations)

−7/del(7q), +8, +9, del(11q), del(20q)


+8, +9, del(11q), del(20q), +21



No specific abnormalities



No recurrent abnormalities


Abbreviations: CML chronic myelogenous leukemia, PV polycythemia vera, PMF primary myelofibrosis, ET essential thrombocythemia, SM systemic mastocytosis, CNL chronic neutrophilic leukemia, CEL, NOS chronic eosinophilic leukemia, not otherwise specified, MPN, U myeloproliferative neoplasm, unclassified

Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a stem cell neoplasm that can occur at any age but is most frequent in the 5th and 6th decades of life [127, 128]. It is characterized by high white blood cell count with increased levels of granulocytes and megakaryocytes, often in the presence of eosinophilia and basophilia. CML is characterized by the t(9;22)(q34;q11.2), which leads to the formation of a chimeric transcript between the ABL1 and BCR genes at 9q34 and 22q11.2, respectively [129, 130] (Fig. 15.4). The derivative chromosome 22 is also known as the Philadelphia (Ph) chromosome and is the first abnormality to have been associated with a specific malignant neoplasm (see also Chap. 1). The Ph chromosome was described in 1960 by Nowell and Hungerford and is named after the city in which it was discovered [4].


Fig. 15.4
Karyogram of a patient with CML in chronic phase. The abnormal chromosomes involved in the t(9;22)(q34;q11.2) are indicated with arrows. The derivative 22 is the Philadelphia (Ph) chromosome

The BCR-ABL1 rearrangement is also the first reported example of a “hybrid” gene leading to the production of an abnormal tyrosine kinase [131, 132]. Three fusion proteins derived from different breakpoints in the BCR gene are known: P210BCR-ABL1, P190BCR-ABL1, and P230BCR-ABL1. The P210BCR-ABL1 is found in the majority of patients with CML and in 30% of patients with Ph-positive acute lymphoblastic leukemia (ALL); the P190BCR-ABL1 is found in about 20% of adults and 80% of children with ALL, in Ph-positive AML, and rarely in CML; and the rare P230BCR-ABL1 is found only in cases of neutrophilic-chronic myeloid leukemia (CML-N), which has been described as a CML variant associated with a more benign clinical course than classic CML [133, 134].

There are three main clinical phases of CML: chronic, accelerated, and blast crisis [135]. The chronic phase of CML is characterized by mild or no symptoms and less than 5% blasts. At this stage, the only abnormality is the t(9;22). About 6% of cases have a variant translocation due to the involvement of one or more additional chromosomes, whereas in approximately 3% of cases the translocation cannot be identified by routine cytogenetics mostly due to cryptic insertions of ABL1 sequences from chromosome 9 into the BCR region on chromosome 22 or vice versa [136, 137]. These variants and cryptic rearrangements generally have the same prognostic outcome of the standard t(9;22), but some are associated with a more aggressive course. This may be due to the fact the variant translocations might be the result of one, two, or more events or they might lead to a deletion of either BCR or ABL1 sequences adjacent to the translocation breakpoints. Fluorescence in situ hybridization (FISH) has revealed small deletions adjacent to the ABL1 and BCR breakpoints in approximately 16 and 8% of cases, respectively [138] (see also Chap. 17).

Conventional cytogenetic analysis can sometimes reveal abnormalities in addition to the t(9;22). It is important to note, however, that an additional balanced rearrangement in all metaphase cells in chronic phase CML (or any neoplasm, for that matter) might be constitutional in origin. This should be investigated and removed from the equation when determined to be the case. When the abnormality in addition to the t(9;22) is obviously (or proven to be) acquired, it is indicative of clonal evolution. At the clinical level, such clonal progression is associated with the accelerated phase or blast crisis, both characterized by an increase in the number of blasts and worsening of clinical symptoms [139]. The most recurrent chromosome abnormalities (about 90% of cases) in these phases are an additional Ph chromosome, +8, i(17)(q10), and/or +19 [140] (Fig. 15.5). Other abnormalities, such as −Y, −7, del(7q), t(8;21)(q22;q22.3), t(15;17)(q24.1;q21.2), inv(16)(p13.1q22.1), as well as 3q21.3, 3q26.2, and 11q23 rearrangements, have been reported but only in a small number of cases.


Fig. 15.5
Karyogram of a patient with CML in blast crisis. This karyogram contains the three most common additional abnormalities observed in the progressive phases of CML, specifically +8, i(17q), and  +  Ph

Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm of adults (50–60 years of age) characterized by a proliferation of red blood cells, which in some patients leads to bleeding and thrombosis [141]. At the chromosome level, patients are BCR-ABL1 fusion-negative, and most, if not all, cases have a mutation at codon 617 in the Janus kinase 2 gene (JAK2, located at 9p24.1) that results in a substitution of phenylalanine for valine (V617F) [60, 142]. Mutations in exons 12 and 13 have also been described in patients negative for JAK2 V617F mutations [143, 144]. Other mutations involving the MPL, TET2, and CBL genes have been found in some of these patients [143, 144]. These mutations are receiving increasing attention, particularly in the area of possible targeted therapy using tyrosine kinase inhibitors. About 20% of cases have an abnormal karyotype at diagnosis, mostly characterized by +8, +9/+9p, and/or del(20q) [98, 145] (Fig. 15.6). Furthermore, gain of 9p is usually the result of a derivative chromosome, the most common of which is a der(9;18)(p10;q10) [146148]. Gain of chromosome 9 or 9p is assumed to represent a gain-of-function mechanism with respect to JAK2 [149]. Less frequently gain of 1q, or partial trisomy 1q, might be seen. This gain is often the result of unbalanced translocations involving chromosome 1 and various chromosome regions [150]. The detection of chromosome abnormalities in PV increases as the disease progresses to MDS or AML [151]. The most common abnormalities during disease progression are del(5q), del(7q), and/or del(17p) [152, 153].


Fig. 15.6
Karyogram of a patient with polycythemia vera. The three most common abnormalities are present in this karyogram, specifically +8, +9, and del(20q)

Primary Myelofibrosis

Primary myelofibrosis (PMF), also known as idiopathic myelofibrosis and agnogenic myeloid metaplasia, is characterized by marrow fibrosis with an increased number of megakaryocytes and immature granulocytes and associated anemia. Affected patients are generally in their 5th and 6th decade of life [154, 155]. Approximately 50% of patients with PMF have the JAK2 V617F mutation, but unlike PV, no mutations of JAK2 other than V617F have been found. A small number of patients have mutations of other genes, particularly MPL [156]. At diagnosis, about 40–50% of cases show chromosome abnormalities, the most common of which are del(13q), del(20q), and gain of chromosome 8 [157, 158]. Additional abnormalities are detected during disease progression, including del(5q), del(7q), gain of 1q, and del(17p) [159].

Essential Thrombocythemia

Essential thrombocythemia (ET) is associated with an increased number of platelets and megakaryocytes, plus fibrosis in the marrow. Patients are generally asymptomatic, with about 50% presenting with circulation problems such as bleeding and thrombosis [160]. Similar to the other classic MPN, JAK2 mutations are also detected in these patients. Approximately 50% have the characteristic JAK2 V617F mutation found in PV and MPF, whereas another 4–5% of patients have mutations of MPL [161]. Only 10% of cases have chromosome abnormalities, which are similar to those seen in PV and PMF. Specifically, +8, +9, del(13q), and del(20q) are the most common, followed by gain of 1q, del(5q), and del(7q) [162]. As in other MPNs, karyotypic abnormalities are more frequent during disease progression to MDS or AML. Because ET is often a diagnosis of exclusion, some clinicians prefer to definitively rule out CML by testing for t(9;22) or a BCR-ABL1 rearrangement in these patients when the karyotype is normal.

Chronic Eosinophilic Leukemia, Not Otherwise Specified (NOS)

Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS), is characterized by hypereosinophilia and represents a rare MPN [163]. The diagnosis is usually achieved by the exclusion of conditions that might be causing the abnormal increase of eosinophils in the marrow and blood. Two entities exist: CEL, not otherwise specified, and CEL with rearrangements involving the platelet-derived growth factor receptors (PDGFRA and PDGFRB) [123, 155]. Pertinent literature indicates that CEL should be distinguished from idiopathic hypereosinophilia by the presence of leukemic blasts. No specific abnormalities have been reported in CEL, NOS. Among the CELs with PDGFR rearrangements, the most common abnormality is deletion of CHIC2 located at 4q12, which leads to a FIP1L1-PDGFRA fusion [164]. See later section: “Myeloid and Lymphoid Neoplasms Associated with PDGFRA, PDGFRB, and FGFR1 .”

Systemic Mastocytosis

Patients with systemic mastocytosis (SM) present with proliferation of mast cells in the bone marrow and/or other organs [165]. Most patients are characterized by symptoms such as hepatomegaly, osteoporosis, and ascites, among others. This is a very complex disease, as it comprises several distinct entities and is also found in association with neoplasms such as MPN and leukemia [165]. The disease course can vary from indolent to aggressive. A large number of cases have rearrangements involving chromosome 4, most likely due to the fact that this disease is often associated with mutations in KIT located at 4q12 [166]. The most common KIT mutation, which results in substitution of valine for asparagine, occurs at amino acid position 816 and is thus known as D816V. This mutation leads to relative resistance to the tyrosine kinase inhibitor imatinib mesylate (Gleevec®) and therefore provides relevant information for treatment selection [167]. Some cases, particularly those associated with hypereosinophilia, present with the same FIP1L1-PDGFRA fusion and other rearrangements involving PDGFRA observed in CEL [168]. Other detectable chromosome abnormalities are similar to those reported for other MPNs and leukemias, specifically +8, +9, del(7q), del(11q), del(20q), t(8;21), and inv(16)/t(16;16). The association of mastocytosis with core binding factors AML, specifically those leukemias with t(8;21) and inv(16)/t(16;16), makes it necessary to investigate these patients for KIT mutations [169].

Chronic Neutrophilic Leukemia

Chronic neutrophilic leukemia (CNL), as the name implies, is characterized by an increase in mature neutrophils [170]. Patients often present with splenomegaly, but no fibrosis is present in the marrow. Approximately 20% of cases have an abnormal karyotype. The abnormalities observed so far include +8, +9, del(11q), del(20q), +21, and less frequently del(12p) [171, 172].

Some CNL patients present with a t(9;22)(q34;q11.2) as seen in typical CML but with a p230 BCR-ABL1 transcript [173]. According to the WHO 2008 classification, these cases should be considered CML with a variant BCR-ABL1 transcript and not CNL.

Myeloid and Lymphoid Neoplasms Associated with PDGFRA, PDGFRB, and FGFR1

This is a rare group of stem cell myeloid and lymphoid neoplasms that have in common the presence of eosinophilia and the involvement of genes that code for a tyrosine kinase [174]. In the WHO 2008 classification, these neoplasms are grouped together under the name, “myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1” [67]. Various translocations involving the PDGFRA (4q12), PDGFRB (5q33.1), and FGFR1 (8p12) genes have been reported (Fig. 15.7). It is essential to clarify that although some earlier publications position the FGFR1 gene locus at 8p11, the present chromosome location following more precise mapping is at 8p12 [175]. The most common translocation observed in these neoplasms is t(5;12)(q33.1;p13.2) leading to a PDGFRB-ETV6 fusion [176, 177]. Some of the rearrangements are cryptic at the chromosome level. Since the presence of translocations involving PDGFRA and PDGFRB is associated with responsiveness to tyrosine kinase inhibitors, it is important, when one of these particular MPNs is suspected, to perform appropriate molecular studies to investigate whether any are present. Some translocations involving 4q12, 5q33.1, or 8p12, but not resulting in a rearrangement of the PDGFRA, PDGFRB, and FGFR1 genes, respectively, have been also reported. In these cases, as well, the final interpretation should be dependent on the presence or absence of the molecular rearrangement. The rearrangement involving PDGFRA and FIP1L1 at 4q12 is cryptic with conventional cytogenetics and can be detected only by FISH or by RT-PCR. However, FISH appears to be superior as it can provide information about other rearrangements involving the 4q12 region [178]. Rearrangements involving PDGFRB, located at 5q33.1, include various translocations, the most common of which is t(5;12)(q33.1;p13.2), which fuses the PDGFRB and the ETV6 genes [179]. FISH is useful and should be performed on these patients since the presence of these rearrangements requires a specific alternative treatment. See Chap. 17, Fig. 17.12a, b and discussion on tyrosine kinases that follows.


Fig. 15.7
Partial karyograms showing some of the most common translocations involving PDGFRA, PDGFRB, and FGFR1. In this particular figure, t(4;12)(q12;p13.2) fuses PDGFRA with ETV6 (a), and t(5;12)(q33;p13.2) fuses PDGFRB with ETV6 (b), whereas t(8;13)(p12;q12) leads to fusion of FGFR1 and FLT3 (c)

Myeloproliferative Neoplasms, Unclassifiable

This category includes stem cell neoplasms that do not have the morphologic characteristics typically seen in any particular MPN [67]. They might have overlapping features seen in various MPNs but nothing specific enough to be classifiable as a specific MPN. Genetically, no rearrangements of PDGFRA, PDGFRB, or FGFR1 are present, and no recurrent chromosome abnormalities have been associated with these neoplasms.

Myeloid Neoplasms with Translocations Involving Genes Coding For Tyrosine Kinases

A number of myeloid neoplasms exhibit translocations involving genes that code for tyrosine kinases other than PDGFRA, PDGFRB, or FGFR1. These neoplasms are not at this time included in a specific group but deserve some consideration, particularly in view of the increasing interest in these genes for therapeutic advancements. See Table 15.7 for a list of these translocations and associated neoplasms. The majority of neoplasms where these translocations have been observed fall into the category of atypical CML (aCML), and the rest have been observed in other myeloid or lymphoid neoplasms [16, 180182].

Table 15.7
Rearrangements involving genes that code for tyrosine kinases and neoplasms associated with them


Gene fusionsa










T-Cell lymphoma







t(9;12)(q34;p13.2) or ins(12;9)(p13.2;q34q34)















Abbreviations: AML acute myeloid leukemia, aCML atypical chronic myeloid leukemia, CEL chronic eosinophilic leukemia, ALL acute lymphoblastic leukemia, MDS myelodysplastic syndrome, MPN myeloproliferative neoplasm

aGenes that code for tyrosine kinases are in bold

Myelodysplastic/Myeloproliferative Neoplasms

This group includes neoplasms with morphologic features that can be seen in both MDS and MPN [183]. Generally, the bone marrow is hypercellular, but there is also some degree of dysplasia. The number of blasts is always below 20%. The neoplasms included here are chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), and myelodysplastic syndrome/myeloproliferative neoplasm, unclassifiable (MDS/MPN, U). Table 15.8 presents some clinical and cytogenetic data for each of these neoplasms. The workup of the diagnosis includes the absence of BCR-ABL1 fusion and of rearrangements of PDGFRA, PDGFRB, and FGFR1. On the other hand, mutations involving transcription factors such as CEBPA, NPM1, or WT1 are frequent in these neoplasms, and one or more of these mutations might be present at the same time. Other significant gene mutations involve TET2, RUNX1, ASXL1, and CBL [184]. The prognosis associated with MDS/MPN is considered, in most cases, unfavorable since these patients rapidly progress to acute leukemia and are generally resistant to chemotherapy with associated short survivals after transformation [183].

Table 15.8
MDS/MPN according to WHO 2008 and most common chromosome abnormalities in order of frequency


Percent of blasts (%)

Recurrent chromosome abnormalities



−7/del(7q), +8, 12p rearrangements, i(17q), del(5q)



+8, del(20q), involvement of chromosomes 12, 13, 14, and 17



−7/del(7q), del(5q)



del(5q) (infrequent)

Abbreviations: CMML chronic myelomonocytic leukemia, aCML atypical chronic myeloid leukemia, JMML juvenile myelomonocytic leukemia, MDS/MPN, U: myelodysplastic syndrome/myeloproliferative neoplasm, unclassified

Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is an MPN characterized by persistent monocytosis and variable degree of dysplasia [185]. The cases that were described previously as having a t(5;12)(q33.1;p13.2) leading to a PDGFRB-ETV6 fusion are now included in the group of neoplasms with rearrangements of PDGFRA, PDGFRB, and FGFR1 [67, 177, 186]. Although no specific abnormality has been associated with CMML, recurrent chromosome abnormalities, such as −7/del(7q), gain of chromosome 8, and less commonly del(5q), 12p rearrangements, and i(17)(q10), have been observed [187189]. See Table 15.8.

Atypical Chronic Myeloid Leukemia

Atypical chronic myeloid leukemia (aCML) is an interesting neoplasm that presents with features seen in classic CML as well as with myelodysplastic characteristics [190]. Although this neoplasm has many similarities with classic CML, it lacks the typical t(9;22)(q34;q11.2). Chromosome abnormalities are detected in the majority of cases and are similar to the ones described for CMML, except for losses involving chromosomes 6 and 7 and i(17)(q10), which seem to be confined to CMML. Thus, gain of chromosome 8 and rearrangements resulting in deletions of 12p are the most frequent aberrations [191, 192]. Furthermore, the t(8;9)(p22;p24) (leading to a PCM1-JAK2 fusion) that was previously associated with aCML is no longer associated with this neoplasm but most likely belongs with chronic neutrophilic leukemia (CNL). In fact, neoplasms with JAK2 mutations should not be considered as aCML [193].

Juvenile Myelomonocytic Leukemia

As the name implies, juvenile myelomonocytic leukemia (JMML) is an MPN of childhood, characterized by an abnormal proliferation of myelocytes and monocytes in the bone marrow [190]. As with the other MPNs in this category, the final diagnosis is based on the exclusion of the BCR-ABL1 fusion [67]. The most common abnormality is −7/del(7q) and less frequently del(5q) [55, 194, 195].

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is defined by the presence of myeloblasts in the bone marrow, peripheral blood, and other tissues [196, 197]. At least 20% blasts should be present in the marrow. However, <20% blasts and presence of a specific/recurrent abnormality associated with a particular subtype of AML is sufficient to make the diagnosis. The classification of these hematologic neoplasms has been revised by the WHO to account for the various genetic and cytogenetic changes that characterize this neoplasm [67]. See Table 15.9 and Fig. 15.8. Although AML more frequently affects adults in their 6th decade of life, it has been described in children and young adults as well [198]. Among the myeloid neoplasms, this is the group that accounts for the majority of specific abnormalities and for a large number of balanced rearrangements, most of which are translocations [39, 199201].

Table 15.9
Acute myeloid leukemia (AML) classification and associated chromosome abnormalities according to the World Health Organization [67] (See Fig. 15.8)


Frequency (%)

Chromosome abnormality (typical and variants)

Common additional abnormalities (in order of frequency)

AML with recurrent genetic abnormalities

AML with t(8;21)



−X or  −  Y, del(9q), del(7q), +8

AML with inv(16) or t(16;16)


inv(16)(p13.1q22.1) or t(16;16)(p13.1;q22.1)

+22, +8, del(7q)

AML with t(15;17)



+8, del(7q), del(9q)

AML with t(9;11)

9–12 (pediatric)


−X or  −  Y, +8

2 (adult)

AML with t(6;9)



+8, +13, +21

AML with inv(3) or t(3;3)


inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)

−7, del(5)

AML (megakaryoblastic) with t(1;22)



del(5q), del(7q), +21

AML with NPM1 mutation


Normal or no specific abnormality

No specific abnormality

AML with FLT3 mutation


Normal or no specific abnormality

No specific abnormality

AML with CEBPA mutation


Normal or no specific abnormality

No specific abnormality

AML with KIT mutation
t(8;21) or inv(16)/t(16;16)

Same are for t(8;21) and inv(16)/t(16;16)

AML with WT1 mutation


Normal or no specific abnormality

No specific abnormality

AML with myelodysplastic-related changes


del(5q), del(7q), +8, del(20q), del(9q)

−X or  −  Y, +1q,

Therapy-related myeloid neoplasms


Complex with del(5q), del(7q), +8, 3q21.3 or 3q26.2 rearrangements, 11q23 rearrangements

del(6p), del(12p), del(17p), del(9q), del(20q), +21

AML, not otherwise specified

AML with minimal differentiation


Complex with del(5q), del(7q), +8, MLL rearrangements, RUNX1 rearrangements

del(17p), del(12p)

AML without maturation


+8, del(9q)

No recurrent abnormality

AML with maturation



No recurrent abnormality

AML (myelomonocytic)



No recurrent abnormality

AML (monoblastic/monocytic)



No recurrent abnormality

AML (erythroid)

Pure erythroid leukemia


Complex with −5/del(5q), −7/del(7q), +8, del(20q)a

del(6p), del(12p), del(17p)

Erythroleukemia (erythroid/myeloid)


Complex with t(3;3)/inv(3), −5/del(5q), −7/del(7q), +8, del(20q)a

del(6p), del(12p), del(17p)

AML (megakaryoblastic)


Children: t(1;22)(p13;q13), +21 Adults: Complex with −5/del(5q), −7/del(7q), t(3;3)/inv(3), +8, del(20q)a

del(6p), del(12p), del(17p)

AML (basophilic)


No recurrent abnormality

No recurrent abnormality

AML (panmyelosis with myelofibrosis)


Complex with −5/del(5q), −7/del(7q), +8, del(20q)a

del(6p), del(12p), del(17p)

Myeloid sarcoma
Abnormalities similar to AML with recurrent genetic abnormalities

Similar to AML with recurrent genetic abnormalities

Myeloid proliferation related to Down syndrome

Down syndrome patients

Transient abnormal myelopoiesis

10 newborns

Additional copies of chromosome 21 (in addition to constitutional trisomy 21)


Myeloid leukemia associated with Down syndrome

1–2 children (<5 years of age)

Additional copies of chromosome 21 (in addition to constitutional trisomy 21)

−7, +8

Blastic plasmacytoid dendritic cell neoplasms


del(4q), del(5q), del(12p), del(13q), del(6q), del(15q), del(9p), del(9q)

No recurrent abnormalities


Fig. 15.8
Partial karyograms showing recurrent (or specific) rearrangements in AML. These translocations/inversions define particular AML subtypes in the WHO classification

Acute Myeloid Leukemia with Recurrent Genetic Abnormalities

The AMLs included in this group are characterized by the presence of well-established genetic abnormalities, the most common of which are t(8;21)(q22;q22.3), inv(16)(p13.1q22.1) or (16;16)(p13.1;q22.1), t(15;17)(q24.1;q21.2), t(9;11)(p22;q23), t(6;9)(p23;q34.1), inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), and t(1;22)(p13.3;q13.1). These translocations/inversions belong to the so-called class 2 mutations, which have the ability to arrest differentiation of the lineage affected by the rearrangement [202]. This results in proliferation of only a particular subset of myeloid cells. As such, these chromosome abnormalities have been associated with particular subtypes of AML.

AML with t(8;21)(q22;q22.3)

This represents one of the core binding factor (CBF) myeloid leukemias and affects approximately 8–10% of AML patients, mainly adults [203]. The t(8;21) leads to a RUNX1-RUNXT1 (formerly AML1-ETO) fusion and is generally associated with a favorable prognostic outcome [46, 47]. This particular AML is also known as AML with maturation and as subtype M2 according to the French-American-British (FAB) classification [204, 205]. Less commonly, t(8;21) can be also seen in AMML (FAB M4) and in therapy-related MDS/AML [206]. Variant translocations, usually affecting a third chromosome, have been reported in 3% of cases [26, 207212]. The presence of additional abnormalities is common (about 70% of cases). The most frequent additional abnormality is loss of a sex chromosome (the Y in males), followed by del(9q), del(7q), +8, and/or +21. Although most of these additional abnormalities do not appear to affect the favorable prognostic outcome associated with t(8;21), gain of chromosome 6 is also seen, and some reports indicate a less favorable disease course when trisomy 6 is part of the karyotype [26, 213].

Regardless of the presence or lack of additional abnormalities, patients exhibit a good response to chemotherapy together with a high rate of complete remission and disease-free survival. However, the favorable prognostic outcome is ­without exception altered by the presence of KIT mutations [214, 215].

AML with inv(16)(p13.1q22.1) or t(16;16)(p13.1;q22.1)

The characteristic of this AML is the presence of myelomonocytic blasts and atypical eosinophils. Also known as AML M4EO according to the FAB classification, this ­leukemia makes up 7–10% of AML cases and is generally associated with a favorable prognostic outcome [203]. However, patients have a higher risk of central nervous system (CNS) involvement at diagnosis or at relapse than patients with other types of AML. Adults are more frequently affected than children. The hallmark of this AML is the inv(16)(p13.1q22.1) or, less commonly, the t(16;16)(p13.1;q22.1). Either abnormality leads to the fusion of MYH11 at 16p13.1 with CBFB at 16q22.1 [216]. The identification of these rearrangements by conventional cytogenetics might be challenging, particularly when the chromosome morphology is not optimal. In those cases, FISH or RT-PCR can be helpful [217]. These rearrangements have been reported occasionally in tMDS and tAML [218]. Chromosome abnormalities in addition to inv(16) or t(16;16) are detected in approximately 30% of cases [219]. The most common is +22, which is considered a clue by many cytogeneticists, particularly when the presence of inv(16) or t(16;16) is not obvious. Other additional chromosome abnormalities include +8, del(7q), and/or +21. Although this leukemia has been associated with complete remission and improved long-term survival, molecular testing for KIT mutations is necessary, as these are associated with adverse prognosis and necessitate more aggressive therapy [214].

Acute Promyelocytic Leukemia with t(15;17)(q24.1;q21.2)

The vast body of research of the past 30 years has contributed to the successful management of acute promyelocytic leukemia (APL) [42, 220]. Originally considered one of the most aggressive leukemias, it is now a model for targeted therapy [221]. Due to the high risk of early death and the potential for high cure rate, it is essential to immediately identify this leukemia. The t(15;17) is the specific abnormality that characterizes this subtype of AML [42]. The formation of this translocation leads to a fusion between PML at 15q24.1 and RARA at 17q21.2 [48]. The PML-RARA fusion is associated with a favorable prognosis and response to treatment with all-trans retinoic acid (ATRA) [220]. Translocations with additional rearrangements involving either chromosome 15 or 17 or complex translocations involving a third chromosome occur in approximately 5% of cases [222, 223]. In these cases, it is important to determine that the PML-RARA fusion is intact. RT-PCR can easily be used to verify this as well as determine the size of transcript, which could negatively influence the prognostic outcome [224]. Other variants involving 17q21.2 and not 15q24.1 exist but are rare. The most known of these variants are t(5;17)(q35.1;q21.2) leading to a fusion of NPM1 and RARA and t(11;17)(q23.2;q21.2) leading to a fusion of ZBTB16 (PLZF) and RARA [225]. The t(5;17) seems to respond to ATRA, whereas t(11;17) does not. The presence of t(15;17) and variants in therapy-related neoplasms is infrequent, but it has been reported [226]. These cases show dysplastic features and often are associated with additional chromosomal and molecular changes.

Additional abnormalities have also been observed in de novo APL, of which +8, del(9q), and del(7q) are the most frequent. The presence of chromosome abnormalities in addition to t(15;17) does not appear to affect the prognosis associated with this neoplasm [227].

AML with t(9;11)(p22;q23) and Other Translocations Involving MLL

This translocation leads to fusion of MLLT3 at 9p22 with MLL at 11q23 and is found in AML with a monocytic or myelomonocytic phenotype, lack of CD34 expression, and frequent RAS mutations [228]. This is the most common translocation involving MLL [67]. Among the approximately 85 known MLL translocations, t(9;11) is thought to be associated with a better prognostic outcome [229]. However, large-scale retrospective studies could not confirm this earlier result [230]. With conventional cytogenetics, MLL translocations are usually present in all or almost all ­metaphase cells analyzed. Additional abnormalities can be seen, and the most common are loss of a sex chromosome (−Y in males) and +8 [231]. Another frequent MLL translocation is t(11;19) with variant breakpoints on chromosome 19. Specifically, the breakpoint at 19p13.1 (ELL) is seen mainly in adults, whereas the breakpoint at 19p13.3 (MLLT1) is typical of childhood AML [232]. t(6;11)(q27;q23) which involves the MLLT4 and MLL genes, respectively, is at times difficult to identify and often has been erroneously identified as del(11q) [233].

Two recurring translocations involving chromosomes 10 and 11 have been observed. The most common involves the MLLT10 gene at 10p12.3. The fusion of this gene with MLL is often the result of an inverted insertion of a variant segment of chromosome 11 containing the 3′ portion of MLL into the short arm of chromosome 10 rather than a reciprocal translocation [234]. The other is t(10;11)(q21.3;q23), which fuses TET1 with MLL.

Due to the cryptic nature of some of these translocations, it is always good practice to perform FISH to look for an MLL rearrangement when the karyotype appears to be normal. See also Chap. 17, Fig. 17.8.

In addition to de novo AML, MLL translocations have also been reported in therapy-related MDS/AML [79] (Fig. 15.9).


Fig. 15.9
Partial karyograms illustrating common MLL (11q23) translocations observed in AML. Some of these translocations have been observed also in therapy-related MDS/AML

AML with t(6;9)(p23;q34.1)

This somewhat rare translocation results in the fusion of DEK at 6p23 with NUP214 at 9q34.1. t(6;9) is probably the abnormality most frequently associated with basophilia and had been seen in both pediatric and adult patients [235]. In most of the cases, this abnormality is present as the only change, but it can also be seen in a complex karyotype, particularly together with gains of chromosomes 8, 13, and/or 21 [236].

AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)

These two abnormalities have in common the involvement of two genes associated with an unfavorable prognostic outcome, RPN1 at 3q21.3 and MECOM (EVI1) at 3q26.2 [103]. There are several other balanced and unbalanced rearrangements involving these two regions, including 1p36, 2p15, 3p12, 3p24, 3q23, 5q31.2, 5q34, 7q21, 8q24, 11p15, 12p13, 12q21, 17q22, 18q11, and 21q22.3 [104106]. Some of these rearrangements are also common in therapy-related MDS/AML. Rearrangements of 3q21.3 and 3q26.2, particularly t(3;21)(q26.2;q22.3), are also seen as additional abnormalities during progression of CML to accelerated phase and blast crisis. The most common additional abnormalities that accompany cases with rearrangements of 3q21.3 and 3q26.2 are −7 and, less frequently, del(5q) [237].

Megakaryoblastic AML with t(1;22)(p13.3;q13.1)

Acute megakaryoblastic leukemia (AMKL; FAB M7) is a clonal stem cell neoplasm that makes up about 3–15% of all AML cases [238]. This leukemia is seen mostly in children, with the median age at presentation between 1 and 8 years [238, 239]. The incidence of developing this subtype of AML is much higher in children with Down syndrome (DS) than in children without DS [240]. Interestingly, DS children generally have a more favorable prognosis compared to patients without constitutional +21 [240].

Three entities of AMKL have been described. The first subtype is observed in Down syndrome (DS) children and is characterized by mutation of GATA1 and also by t(1;22)(p13.3;q13.1), leading to a fusion of RBM15 at 1p13.3 with MKL1 at 22q13.1 [241]. GATA1 mutations are rare in ­non-DS AMKL [242]. The few non-DS cases with GATA1 mutation are characterized by the presence of an acquired trisomy 21 in the karyotype [242]. This is intriguing and raises the possibility that GATA1 mutation might be dependent on the presence of an additional copy of chromosomes 21. The second subtype is observed in about 20% of infants with Down syndrome and transient myeloproliferative disease (TMD) who subsequently develop AMKL [240]. GATA1 is likely to play a critical role in the etiology of TMD and mutation of this gene represents a very early event in the development of AMKL. The karyotype of these DS patients typically contains additional copies of chromosome 21 (four or more copies of chromosome 21 can be seen), as well as gain of chromosome 8. The third subtype of AMKL is found in infants that show the t(1;22) but do not have Down syndrome [241, 242]. Detection of the t(1;22) is diagnostic in this group. The prognosis associated with the t(1;22) used to be considered unfavorable but is now considered intermediate since these patients are responsive to AML therapy and exhibit long clinical remission times.

In adults, this leukemia is often secondary in nature, either posttreatment or during leukemic transformation [239]. Approximately 50% of adult patients have chromosome abnormalities at diagnosis. The most common rearrangements involve the regions 3q21.3 and 3q26.2. In addition, frequently, the abnormal karyotype includes −5/del(5q), −7/del(7q), and +8 [238]. t(1;22) has not been observed in adults.

Acute Myeloid Leukemia with Gene Mutations

This new category of AML is characterized by a normal karyotype and recurrent gene mutations involving genes such as nucleophosmin (NPM1) located at 5q35.1, fms-like tyrosine kinase 3 (FLT3) located at 13q12.2, CCAAT/enhancer-binding protein-α(alpha) (CEBPA) located at 19q13.1, and mixed lineage leukemia (MLL) located at 11q23 [243, 244]. Specifically, 45–60% patients have mutations involving NPM1, 20–35% patients show mutations of FLT3, 10–20% have CEBPA mutations, and 5–25% have MLL tandem duplications. Some of these mutations are not mutually exclusive; most notably, NPM1 and FLT3 might be present at the same time. The prognosis is variable and depends on which gene is mutated. Patients with NPM1 or biallelic CEBPA mutation alone have a favorable prognosis, whereas the presence of FLT3 or MLL mutations is associated with an unfavorable prognostic outcome [245]. Therefore, patients with both FLT3 and NPM1 mutations have an adverse prognosis. Other less frequent mutations observed in AML with normal or abnormal karyotype involve KIT, WT1, KRAS, and NRAS. Of note, KIT is also associated with abnormalities involving the core binding factor genes, such as t(8;21)(q22;q22.3) and inv(16)(p13.1q22.1)/t(16;16)(p13.1;q22.1) [214]. Since KIT mutations affect the clinical course, a suggestion to investigate for the presence of KIT mutations should be provided for patients characterized by one of these chromosome abnormalities.

Acute Myeloid Leukemia with Myelodysplasia-Related Changes

As the name implies, these are myeloid leukemias characterized by abnormalities typically seen in MDS, specifically −5/del(5q), −7/del(7q), and +8, as well as translocations involving 3q21.3, 3q26.2, and 11q23. The majority of these AML have complex karyotypes similar to what has been reported in high-grade MDS and tMDS/tAML. However, according to the WHO, this group should not include patients that have a prior history of cytotoxic or radiation therapy [246].

Myeloid Sarcoma

Myeloid sarcoma or granulocytic sarcoma is the name given to a myeloid leukemic process that forms a mass at an anatomical site outside of the bone marrow; the term extramedullary myeloid tumor is therefore also used to describe this leukemia [247]. Other terms used include granulocytic sarcoma and chloroma. This leukemia occurs at any age and affects males more than females. It can arise de novo, represent a relapse of a known leukemia, or occur as a transformation of a chronic myeloproliferative neoplasm or myelodysplastic syndrome.

Several cytogenetic abnormalities have been observed in myeloid sarcoma. The most common are −7, +8, del(5q), del(20q), +4, +11, del(16q), inv(16)/t(16;16), MLL rearrangements, and t(8;21)(q22;q22.3) [248250]. The prognosis is variable as it is influenced by several factors including but not limited to age, morphology, and cytogenetic abnormality.

Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm is a very aggressive leukemia derived from the plasmacytoid monocytes and usually involves the skin, bone marrow, and peripheral blood. This neoplasm is best known as blastic natural killer lymphoma [251]. The median survival is around 12 months. Patients are typically in their sixth decade of life at presentation. Around 20% of cases transform into acute myeloid leukemia, preferentially acute myelomonocytic leukemia. The majority of cases have an abnormal karyotype that is usually complex. The most common abnormalities include del(5q), del(12p), del(13q), del(6q), del(15q), del(4q), del(9p), and del(9q) [252].

Acute Leukemia of Ambiguous Lineage

This group of neoplasms includes acute undifferentiated leukemia (AUL) and mixed phenotype acute leukemia (MPAL) that have not differentiated into a particular lineage or expressed cell surface markers of more than one lineage, respectively [253]. In other words, AUL blasts express neither lymphoid nor myeloid markers, whereas MPAL blasts express markers of different lineages. Although no specific or recurrent abnormalities are observed in AUL, MPAL is characterized by two recurrent abnormalities, t(9;22)(q34;q11.2) and 11q32 (MLL) rearrangements [254]. The prognosis associated with these leukemias is poor. However, patients characterized by the BCRABL1 fusion are expected to have a better course due to response to imatinib.

Lymphoid Neoplasms

This group of hematologic neoplasms includes immature and mature neoplasms of B-cell, T-cell, and natural killer (NK) cell subtypes. Neoplasms of B-cell origin are more frequent than those of T-cell origin [255]. Immature B-cell neoplasms include precursor B-cell lymphoblastic leukemia/lymphoma (pre-B-ALL/LBL) and precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-ALL/LBL) [256]. The yearly incidence of these immature neoplasms is estimated to be 1–4.75/100,000 individuals worldwide. They are by far more common in children than adults. Approximately 85% are of B-cell origin and present as ALL, whereas precursor T-cell lymphoblastic neoplasms present mostly as lymphoma and affect mainly adolescent males.

Disorders of mature cells make up 90% of all lymphoid neoplasms [255]. These lymphomas are more frequent in developed countries with 33 cases/100,000 individuals diagnosed each year.

The majority of lymphoid neoplasms (both precursor and mature types) are characterized by recurrent chromosome abnormalities. Some of the most common subtypes are discussed as follows.

Acute Lymphoid Neoplasms

Acute B-cell Lymphoblastic Leukemia/Lymphoma

This neoplasm is defined as leukemia when it involves the bone marrow and peripheral blood and as lymphoma when it presents as a lesion without evidence of bone marrow and peripheral blood involvement [256]. There is often extramedullary involvement, particularly of the central nervous system, lymph nodes, spleen, liver, and testis in cases of B-ALL and of skin, soft tissue, bone, and lymph nodes in cases of LBL. A large percentage of ALL cases, especially those involving children, are classified as precursor B-cell ALL (pre-B-ALL).

Several factors impact the prognosis. Approximately 85% of B-ALL patients are children [257]. In general, older age (≥10 years) and high WBC are factors associated with high-risk B-ALL, compared with younger age and low WBC, which are associated with low-risk disease [256]. Chromosome abnormalities have been reported in the majority of cases and are useful for prognostic stratification [258, 259]; conventional metaphase cytogenetics is still considered the basic method for the detection of these abnormalities. Pediatric cases with t(9;22)(q34;q11.2), 11q23 (MLL) rearrangements, t(1;19)(q23.3;p13.3), and hypodiploidy (≤45 chromosomes) are known to have an unfavorable prognosis, whereas t(12;21)(p13.2;q22.3) and hyperdiploidy (>50 chromosomes) are associated with a favorable prognostic outcome particularly if trisomies 4 and 10 are present in the latter [258] (see Table 15.10). Cytogenetic and FISH analyses are indicated for proper risk stratification.

Table 15.10
Recurrent chromosome abnormalities and involved genes in B-ALL

Cytogenetic abnormality

Gene(s) involved

Common additional abnormalities


% of patients



+der(22)t(9;22), –7

High risk

2.5% children

25% adults



del(6q), del(11q), 12p rearrangements, del(16q), +21

Low risk

30% children

Absent in adults

Hyperdiploidy (≥50 chromosomes)


Rare structural rearrangements

Low risk

25% children

5% adults

Hypodiploidy (≤45 chromosomes)


Few structural rearrangements

High risk

2% children and teenagers



dup(1q), del(6q), +8, i(9q), i(17q), +21

High risk

Children: 25% pre-B-ALL and 5% B-ALL

Adults: 3% pre-B-ALL



del(6q), del(12p)


10% children and adults

RUNX1 amplification


Generally none

High risk

5% children

2% adults

11q23 rearrangements, including partial deletions and duplications


Generally none

High risk

80% infants

10% children and adults

Children and young adults (generally up to 21 years of age) enrolled in the Children’s Oncology Group (COG) program are required to have their bone marrow or informative peripheral blood sample analyzed by conventional cytogenetics and FISH [260]. The latter is mainly geared toward the detection of prognostic markers such as BCR-ABL1 and ETV6-RUNX1 fusions, MLL rearrangements (including partial deletions/duplications), as well as trisomies 4 and 10. Some of these recurrent abnormalities are discussed at length as follows. Other FISH probes are available to detect and/or clarify less common, atypical, or prognostically less informative chromosome abnormalities. See also Chap. 17.

The Philadelphia Chromosome

The Philadelphia (Ph) chromosome derived from the t(9;22)(q34;q11.2) occurs in approximately 2.5% of children and approximately 25% of adults with B-ALL [261]. At the molecular level, the breakpoints in B-ALL and CML differ, and this variation leads to the production of p190 and p210 fusion proteins, respectively. Approximately 20% of Ph-positive B-ALL patients, however, have been found to generate both the p190 and p210 fusion transcripts, possibly as a result of alternative splicing or missplicing events in the BCR gene [262, 263]. Alternatives to the typical translocation include insertions of ABL1 into the BCR locus and vice versa to form the BCR-ABL1 fusion [264, 265]. Some of these variants and most of the insertions will not result in a classic Ph chromosome with conventional chromosome analysis. However, the presence of the gene fusion will be revealed by FISH and/or PCR testing.

Chromosome abnormalities in addition to the Ph chromosome are seen in greater than 60% of patients and are similar to those observed in CML during progression to accelerated phase or blast crisis, specifically +8 and one extra copy of the Ph chromosome [266]. However, i(17)(q10) is seen primarily in CML, while −7, +X, and del(9p) are seen primarily in B-ALL [267] (Fig. 15.10). While additional abnormalities are associated with disease progression in CML, they do not appear to modify the disease’s course in B-ALL. However, patients with loss of chromosome 7 seem to have a much worse prognosis than patients without this abnormality, probably due to its association with resistance to therapy [268, 269].


Fig. 15.10
Karyogram of a patient with B-ALL. The t(9;22)(q34;q11.2) is rarely the sole abnormality in B-ALL. In this case, there is also loss of one copy of chromosome 7

MLL Rearrangements

Rearrangements involving MLL at 11q23 have been reported in infants, children, and adults with B-ALL [228, 268, 269]. They have been observed in approximately 80% of infants (<1 year old) and 5–10% of children and adults with B-ALL. In children, the cells have a pre-B immunophenotype that express myeloid antigens and are CD19+/CD10− by flow cytometry. The CD10− immunophenotype, high WBC, and young age are helpful clues suggestive of the presence of an MLL rearrangement.

The most frequent MLL translocations include t(4;11)(q21.3;q23) leading to MLL-AFF1 fusion and t(11;19)(q23;p13.3) leading to MLL-MLLT1 fusion [15, 270, 271]. Occasionally, t(9;11)(p22;q23) involving MLLT3 and MLL or other less frequent translocations might be seen (Fig. 15.11). MLL rearrangements are associated with an unfavorable prognostic outcome in both children (particularly infants) and adults, and bone marrow transplant is still the treatment of choice.


Fig. 15.11
Partial karyograms illustrating some of the most recurrent MLL (11q23) translocations in B-ALL. These are presented in order of frequency. The majority of patients with MLL rearrangements are infants

MLL rearrangements have also been reported in T-ALL [272]. Even though these rearrangements are uncommon, recent studies indicate that MLL rearrangements are one of the early leukemogenic hits in T-ALL [273].


Approximately 5% of children with pre-B-ALL and 5% of children with B-ALL have a t(1;19)(q23.3;p13.3), which leads to a fusion of TCF3 located at 19p13,3 with PBX1 at 1q23.3 [274]. This translocation is rare (3% of cases) in adults, who also present with a pre-B immunophenotype. The majority of patients (75% of cases) have an unbalanced form of the translocation, der(19)t(1;19)(q23.3;p13.3), whereas 25% have the balanced t(1;19). Whether the unbalanced form in pediatric B-ALL patients is associated with a better prognostic outcome than the balanced t(1;19) is still controversial [275]. A variant form of t(1;19) is t(17;19)(q22;p13.3), which leads to a fusion between the HLF and TCF3 genes located at 17q22 and 19p13.3, respectively, and has been observed in approximately 1% of pediatric B-ALL patients. This variant translocation is associated with poor prognostic outcome [276]. Both t(1;19) and t(17;19) are easily identifiable with conventional cytogenetics. However, since cells with these abnormalities are characterized by low mitotic activity in culture, their detection might require the analysis of more than the 20 metaphase cells typically examined. Probes that target both TCF3 (break-apart probe) and the actual TCT3-PBX1 fusion are available for initial detection and monitoring [277]. These FISH studies have also proven useful to detect cryptic rearrangements such as inv(19)(p13.3q13.4), which leads to a fusion of the TCF3 and TFPT genes. This inversion has been reported in approximately 5% of pediatric B-ALL cases [274].


Hypodiploidy is associated with an unfavorable prognosis. Fortunately, only 2% of pediatric patients with B-ALL and rare cases of adult patients are found to have a hypodiploid chromosome complement [278280].

Three separate groups have been observed. The most common is the near-haploid karyotype, with a chromosome count ranging from 26 to 29 [281]. The loss and retention of chromosomes in this group is not random. In fact, invariably, the karyotypes with 26 chromosomes retain two copies of chromosomes 14, 21, and the sex chromosomes, with a single copy of all other chromosomes. The chromosomes that are preferentially lost include chromosomes 3, 7, 15, and 17 [281]. Therefore, the investigation of hypodiploidy by FISH should target regions on the preferentially lost chromosomes. The second and third groups include karyotypes with a chromosome count ranging from 30 to 39 and 40 to 44, respectively. Generally, a lower number of chromosomes correspond to a worse prognosis. A peculiarity of hypodiploid karyotypes is their tendency to double the chromosome complement via endoreduplication. This is not a culture-induced (in vitro) doubling, but rather it occurs in vivo, as demonstrated by DNA index studies of uncultured specimens [282] (Fig. 15.12a, b). These studies have shown the presence of three distinct populations: a hypodiploid complement with DNA index below 1, a diploid complement with DNA index equal to 1, and a hyperdiploid complement with DNA index above 1 [283].


Fig. 15.12
Karyograms of a patient with B-ALL showing (a) a near-haploid karyotype and (b) its doubling version. Note the retention of chromosomes 14, 21, and the sex chromosomes. This is the one of the most basic doubling, from which it is easy to suspect the presence of the hypodiploid counterpart

From the DNA index, it is possible to estimate the number of chromosomes present in the karyotype by using the simple mathematical formula 46  ×  DNA index  =  number of chromosomes in the karyotype. The reason(s) that cells with a hypodiploid complement endoreduplicate is not clear. Some authors have suggested that hypodiploid cells are unstable and doubling their complement gives them the ability to survive longer. This might be the reason why, with conventional cytogenetics, more cells with the doubled complement than with the hypodiploid complement are observed. It is imperative to distinguish a doubling from a true hyperdiploid clone since they are associated with different prognostic outcomes. Molecular testing, such as microsatellite markers and SNP array, could be useful to distinguish true hyperdiploid from an endoreduplicated hypodiploid cell population.


True hyperdiploidy (51–68 chromosomes) occurs in approximately 25% of children and 5% of adults with B-ALL [282]. A relatively young age (2–10 years) is associated with a favorable prognosis; children also tend to present with favorable features such as low WBC and a pre-B immunophenotype. Modal chromosome numbers between 51 and 55 are thought to be associated with a relatively less favorable prognosis than those from 56 to 68 chromosomes [259]. The better prognosis of the latter seems to correlate with the presence of trisomies 4 and 10. The most common gains involve chromosomes 4, 6, 8, 10, 14, 17, 18, 19, and 21 (Fig. 15.13). Gain of chromosome 21 (often tetrasomy 21) is the most common numerical abnormality in B-ALL, present in more than 95% of cases with hyperdiploidy. This is followed by gains of chromosomes 6 (85% of cases, especially in adults). X and 14 (80% of cases), 17 and 18 (70% of cases), and 10 (55% of cases). For reasons that are not completely clear, the prognostic outcome of adult B-ALL patients with hyperdiploidy is not as favorable as in children.


Fig. 15.13
Hyperdiploid karyogram of a patient with B-ALL. Chromosomes X, 4, 10, 14, 17, 18, and 21 are usually overrepresented. The best prognosis is associated with the presence of trisomies 4 and 10, as seen here

High hyperdiploidy is sometimes associated with the presence of poor prognostic markers such as t(9;22) and t(1;19) [284]. Another hyperdiploid group with 47–50 chromosomes has been described in approximately 10–15% of children and 2–5% of adults with B-ALL [259]. Trisomy 21 is again the most common numerical abnormality. Furthermore, patients in this group often also exhibit structural chromosome rearrangements [285].


Prior to the discovery of the cryptic t(12;21), which fuses ETV6 at 12p13.2 with RUNX1 at 21q22.3, at least 30% of B-ALL cases were thought to have a normal or prognostically informative karyotype [286]. This translocation is most often seen in children between 2 and 12 years old, with a peak at 3–5 years of age. These children have disease that is characterized by a long duration of first remission and excellent cure rates. t(12;21) is rare in adults with an incidence of 2–5% of cases.

After the first detection of the ETV6-RUNX1 fusion by FISH, a large number of studies demonstrated that the t(12;21) is rarely the only abnormality present. Additional abnormalities include del(6q), del(11q), rearrangements of 12p, and del(16q), and often these abnormalities provide a clue that a t(12;21) might be present [287, 288] (Fig. 15.14).


Fig. 15.14
Karyogram of a patient with B-ALL and cryptic t(12;21). Even if the translocation is cryptic at the conventional cytogenetic level, additional abnormalities, in this case deletions of 6q and 11q (arrows), often serve as a clue

Variant ETV6-RUNX1 fusion patterns can be seen, as demonstrated by FISH studies. The most common of these variants is loss of the native ETV6 allele, generally subsequent to a translocation or other rearrangement involving 12p [289]. Molecular studies have demonstrated that fusion with ETV6 converts RUNX1 from an activator to a repressor of transcription [290]. Molecular studies have also demonstrated that the presence of t(12;21) occurs early and is most likely present in utero. Using PCR, researchers confirmed the presence of t(12;21) in cord blood and perinatally obtained blood samples (Guthrie cards) of patients who later developed t(12;21)-associated ALL. Furthermore, these studies demonstrated the presence of both the typical and several variant ETV6-RUNX1 fusion patterns at levels higher than what is seen in overt B-ALL [291, 292]. This implies that the t(12;21)-carrying cells present in those early samples most likely represent clones predisposed to leukemia development and that the acquisition of more genetic aberrations is needed for one of these clones to become fully malignant.

The usefulness of ETV6-RUNX1 FISH studies goes beyond the ability to merely detect the fusion. Two additional abnormalities in particular can be detected with the ETV6/RUNX1 FISH probes—deletions of ETV6 and amplification of RUNX1. Specifically, a small group of B-ALL patients with a median age of 9 years have been found to have amplification of RUNX1, defined as multiple copies of this gene clustered in a marker chromosome. In the majority of the cases, this amplification is actually concentrated on an abnormal chromosome 21 (iAMP21) [293]. This abnormality is associated with an unfavorable prognosis characterized by high risk of relapse and a decreased event-free and overall survival at 5 years.

Rearrangements of 9p

Unbalanced rearrangements of the short arm of chromosome 9, usually leading to loss of 9p, have been observed in various neoplasms and are particularly frequent in ALL of both B- and T-lineages [294, 295]. In B-ALL, these deletions have been observed in approximately 10% of patients and often are not easily detected with conventional ­cytogenetics. Therefore, FISH testing targeting the CDKN2A gene located at 9p21.3 is very helpful to demonstrate the deletion [296]. Homozygous deletions of this gene, frequently unsuspected, are also confirmed by FISH. Although the CDKN2A gene is thought to play a key role, other genes located at 9p, such as MLLT3, PAX5, MTAP, IFN, JAK2, and PTPLAD2, may play important roles as well [296]. In adults with B-ALL, the presence of del(9p) appears to be associated with improved outcome, whereas in children the same deletion is associated with poor outcome [297]. Besides pure deletions, other unbalanced rearrangements include dic(9;20)(p13.2;q11.2), dic(9;12)(p13.2;p12.2), and i(9)(q10), all of which are associated with an excellent prognostic outcome [298].

This being a textbook dedicated to cytogenetics, it is important to mention some of the “tricks of the trade.” One of these involves the apparent loss of chromosome 20 in some of the karyotypes of patients with B-ALL. FISH has shown that the apparent loss of chromosome 20 is actually the result of a dic(9;20)(p13.2;q11.2). This abnormality, which results in loss of most of 9p and 20q, might be difficult to detect in a sample with poor chromosome morphology, and the apparent loss of chromosome 20 can provide a helpful hint [299] (Fig. 15.15).


Fig. 15.15
Karyogram of a patient with B-ALL showing apparent loss of chromosome 20. Upon careful review, it is possible to recognize that the right chromosome 9 is in reality a dic(9;20)(p13.2;q11.2)

Rearrangements of 14q32.3 (IGH@)

Most of the IGH@ rearrangements observed in B-ALL are balanced translocations. The most common are t(8;14)(q11.2;q32.3), inv(14)(q11.2q32.3), t(14;14)(q11.2;q32.3), t(14;19)(q32.3;q13.1), and t(14;20)(q32.3;q13.1) [300302]. These translocations appear to be more frequent in adults (approximately 10% of cases) than in children (approximately 2% of cases) with B-ALL.

These recurrent IGH@ translocations have in common the deregulated expression of unmutated CEBP genes (CCAAT enhancer-binding protein transcription factors) [303]. A rare t(5;14)(q31.1;q32.3), which leads to an IL3-IGH@ fusion, has also been observed in B-ALL and is often associated with eosinophilia [304, 305]. Other reported IGH@ translocations include t(6;14)(p22.3;q32.3) and t(9;14)(p13.2;q32.3), leading to a fusion of IGH@ with ID4 and PAX5, respectively [306, 307].

Rarely, deletions involving IGH@ have been reported. Based on the small number of cases reported so far with this abnormality, the prognosis is unknown [308].

Two cryptic translocations, t(X;14)(p22.3;q32.3) and t(Y;14)(p11.3;q32.3), have recently been described in B-ALL, especially in patients with Down syndrome. These translocations lead to overexpression of the cytokine receptor gene, CRLF2, located in the X/Y pseudoautosomal region, via juxtaposition of this gene to the IGH@ enhancer. This deregulation can also arise via a cryptic interstitial deletion within the pseudoautosomal region, that is, del(X)(p22.33p22.33)/del(Y)(p11.32p11.32) via juxtaposition of CRLF2 to the P2RY8 promoter also located in the X/Y pseudoautosomal region. The deregulation of CRLF2 is associated with activating mutations in JAK2 [309]. Therefore, performing FISH with an IGH@ probe is a valuable option, particularly in Down syndrome patients with a karyotype lacking acquired chromosome abnormalities.

Acute T-Cell Lymphoblastic Leukemia

Approximately 15% of children and 25% of adults diagnosed yearly with ALL have T-cell ALL/LBL [67]. There appears to be a prevalence of adolescent males (age range 12–19 years), but this is by no means exclusive [310]. Patients with this leukemia often present with mediastinal mass, CNS involvement, and leukocytosis [256].

The cytogenetic abnormalities most often found in T-ALL involve the T-cell receptors, specifically TRA@ and TRD@ at 14q11.2, TRB@ at 7q35, and TRG@ at 7p14 [311]. TRD@ is contained within the TRA@ locus, and thus TRA@ is commonly used as reference.

Several translocations involving these genes have been reported; some are cryptic with conventional cytogenetics [312]. Except for the Y chromosome, virtually every chromosome has been involved with one or more of these T-cell receptors. The most common translocations involve chromosomes 1, 7, 9, 10, 11, 12, and 14 [313] (see Table 15.11).

Table 15.11
Recurrent chromosome abnormalities and involved genes in T-ALL



% Children

% Adults









t(5;14)(q35.1;q32.2) (cryptic)








inv(7)(p15.2q34) or t(7;7)





























del(9)(p21.3) (homozygous/hemizygous)




80 by FISH

















12p rearrangements

ETV6, others



inv(14)(q11.2q32.1) or t(14;14)(q11.2;q32.1)




A rare but recurrent abnormality seen in T-ALL is inv(14)(q11.2q32.1) or t(14;14)(q11.2;q32.1). Either of these rearrangements generally leads to overexpression of TCL1A/TCL1B at 14q32.1 via relocation to the TRA@/TRD@ locus at 14q11.2. TCL1A and TCL1B have approximately 65% homology, and therefore overexpression of one affects the other [314]. Other genes in the vicinity of TCL1A/TCL1B, such as BCL11B at 4q32.3, can also be involved, and thus overexpressed, as a result of this inversion [315]. The prognosis associated with this abnormality is unknown.

Another frequent abnormality is deletion of 9p21.3 leading to loss of CDKN2A. This gene encodes the p14ARF protein, which binds to and inactivates HDM-2. HDM-2 in turn targets the TP53 tumor suppressor protein for degradation [316]. Therefore, deletions of 9p21.3 result in reduction in the amount of p14ARF, loss of inhibition of HDM-2, and subsequent inhibition of TP53 protein production. This deletion can be seen in about 30% of cases by conventional cytogenetics and about 80% of cases with FISH [317]. This indicates that the majority of deletions involving 9p are cryptic. Approximately 50% of these deletions affect both chromosome 9 homologs. This deletion is less frequent (8–10%) in adults with T-ALL.

A relatively new abnormality, discovered by chance while investigating the frequency of BCR-ABL1 fusion in T-ALL, is the amplification of ABL1 [318, 319]. The rearrangement is a cryptic episomal NUP214-ABL1 translocation and occurs in approximately 6% of patients, most of whom are children. FISH with the BCR-ABL1 probes and RT-PCR can both detect T-ALL patients with ABL1 amplification. The quick identification of this rearrangement is fundamental in the clinic because this T-ALL subset is Gleevec® sensitive but may become resistant due to the development of additional mutations (9p21.3 deletions often accompany this amplification) [320]. ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.

Mature B-Cell Neoplasms

Culturing of Mature B-Cell Neoplasms for Cytogenetic Analysis

The detection of chromosome abnormalities in mature B-cell lymphomas by conventional cytogenetics is dependent upon the culturing method used. Historically, the utilization of B-cell mitogens has proven effective in promoting the growth of the abnormal clonal population in culture [321, 322]. The recent introduction of CpG-oligodeoxynucleotides (CpG-ODNs) has increased the detection of clonal abnormalities. CpG-ODNs are made of short single-stranded DNA, are known to activate cells of the immune system in a sequence-dependent manner, and are also known to promote proliferation of chronic lymphocytic leukemia (CLL) cells [323]. One of these CpG-ODNs is DSP-30. In combination with interleukin-2 (IL-2), DSP-30 has proven to be effective in increasing the detection of chromosome abnormalities with conventional cytogenetics when compared to other traditional well-established B-cell mitogens, not only in CLL but also in other mature B-cell lymphoid neoplasms [8, 324, 325]. Table 15.12 shows the author’s experience using the DSP-30/IL-2 cocktail. Table 15.13 lists recurrent chromosome abnormalities in B-cell neoplasms.

Table 15.12
Neoplasms stimulated with DSP-30/IL-2 and associated cytogenetic abnormality rates. A. Meloni-Ehrig, personal data


Number of cases

Abnormal cases

Normal cases

% abnormal

















































































Abbreviations: BL Burkitt lymphoma, CLL chronic lymphocytic leukemia, v-CLL variant type, DLBCL diffuse large B-cell lymphoma, FL follicular lymphoma, HCL hairy cell leukemia, HL Hodgkin lymphoma, LPD lymphoproliferative disorder, MCL mantle cell lymphoma, b-MCL mantle cell lymphoma, blastoid type, MALT mucosa-associated lymphoid tissue, sMZBCL marginal zone B-cell lymphoma, splenic type, NHL non-Hodgkin lymphoma, B-PLL B-cell prolymphocytic leukemia, LPL lymphoplasmacytic lymphoma

aDefined according to immunophenotypic and/or morphologic analyses

bNo specific subtype

Table 15.13
Mature B-Cell lymphoid neoplasms and associated recurrent chromosome abnormalities, according to the World Health Organization

Mature B-cell neoplasm

Primary chromosome abnormalities

Chromosome abnormalities during progression


del(6q), del(11q), del(13q), +12, del(17p), t(2;14), t(14;19), t(14;18),

del(14q),+18, t(8;14) or variants, 13q rearrangements


del(6q). del(11q), +12, del(13q), del(17p)

MYC translocations


t(11;14) and other CCND1 variants; t(6;14) and CCND3 variants; t(12;14) and CCND2 variants

Gains of chromosomes 3, 8, and 15q; losses of 1p, 8p, 9p, 11q, and 13q.

Splenic MZBCL

del(7q); +3, +12, +18



+12, +18, 3q27 rearrangements



t(11;18) or t(14;18); +3 with or without +18; t(1;14) and variants; (3;14)



+5, del(6q), del(7q), +12, del(17p)



del(6q), +4, +3, +7



t(14;18) and variants

Gains of chromosomes X, 2, 7, 8, and 12; del(1p), del(1q), del(6q), del(10q), +der(18), del(22q)


t(14;18), 3q27 rearrangements

1q and 14q rearrangements, del(6q), del(10q), del(11q), del(13q), del(17p), +X, +7, +12, +18


t(8;14) and variants

Gain of 1q


High risk: hypodiploidy, 1p/1q rearrangements, del(13q), t(4;14), t(14;16), t(14;20), del(17p)

del(4q), del(6q), del(16q), del(20q), MYC translocations
Standard risk: hyperdiploidy with gain of odd number chromosomes (+5, +9, +11, +15), without high-risk markers; t(11;14)


Similar to high-risk PCM: hyperdiploidy with gain of odd number chromosomes (+5, +9, +11, +15) and high-risk markers [rearrangements of chromosome 1, del(13q), del(17p)]

MYC translocations


t(8;14) and variants, t(14;18) and/or 3q27 rearrangements, and/or t(11;14)

Same as DLBCL


3q27 rearrangements, del(17p)



Hyperdiploidy, del(1p), del(6q), del(7q), del(13q), del(16q), del(17p), gain of 2p, 9p, +12, rearrangements of 3q27


Abbreviations: CLL chronic lymphocytic leukemia, SLL small lymphocytic lymphoma, PLL prolymphocytic leukemia, MCL mantle cell lymphoma, MZBCL marginal zone B-cell lymphoma, MALT mucosa-associated lymphoid tissue, HCL hairy cell leukemia, LPL lymphoplasmacytic lymphoma, FL follicular lymphoma, DLBCL diffuse large B-cell lymphoma, BL Burkitt lymphoma, PCM plasma cell myeloma, PBL plasmablastic lymphoma, HL Hodgkin lymphoma

Non-Hodgkin Lymphoma

Non-Hodgkin lymphoma (NHL) comprises a heterogeneous group of disorders characterized by localized proliferation of lymphocytes. The WHO recognizes that genetic anomalies represent one of the most reliable criteria for classification of malignant lymphomas [67]. Some tend to be confined to a particular lymphoma—for example, t(14;18)(q32.3;q21.3) in follicular lymphoma—whereas others are nonspecific and can be seen in a variety of lymphomas, such as del(6q) and del(14q).

Most NHL cases are of B-cell origin and are characterized by rearrangements involving the immunoglobulin genes: IGH@ at 14q32.3, IGK@ at 2p12, and IGL@ at 22q11.2 [321]. Translocations can be simple or complex and at times have partial deletions or partial duplications of the genes involved in the translocation. By far, the majority of translocations involve IGH@ [36, 37].

The most common associations between chromosome anomalies and specific lymphomas include t(14;18)(q32.3;q21.3) and follicular lymphoma (FL), t(8;14)(q24.2;q32.3) and Burkitt lymphoma (BL), t(11;14)(q13;q32.3) and mantle cell lymphoma (MCL), and t(11;18)(q21;q21.3) and mucosa-associated lymphoid tissue (MALT) lymphoma [37].

Follicular Lymphoma

Follicular lymphoma (FL) affects 1 in 24,000 individuals per year in the USA [322]. The majority of patients have an indolent disease, and few develop a more aggressive form [326]. Approximately 85–90% of patients with FL and 25–30% of patients with diffuse large B-cell lymphoma (DLBCL) exhibit the t(14;18)(q32.3;q21.3), which results in fusion of BCL2 at 18q21.3 and IGH@ at14q32.3 [327] (Fig. 15.16a). This translocation, which is one of the most common abnormalities in NHL, repositions the BCL2 oncogene so that it is under the control of IGH@ promoter, leading to overexpression of BCL2 and therefore overproduction of BCL2, one of the proteins involved in regulation of apoptosis [52]. Variant translocations, such as t(2;18)(p12;q21.3) and t(18;22)(q21.3;q11.2) involving IGK@ at 2p12 or IGL@ at 22q11.2, respectively, have been described in both FL and DLBCL [328]. These translocations also lead to the overexpression of BCL2.


Fig. 15.16
(a) Karyogram of a patient with follicular lymphoma showing t(14;18)(q32.3;q21.3) as the sole abnormality and (b) together with other rearrangements in a patient in progression to diffuse large B-cell lymphoma. Typical abnormalities during progression are the gain of chromosomes X, 7, and the der(18), as seen in this case

Numerous additional chromosome abnormalities are identified by conventional cytogenetics. In addition to t(14;18), certain numerical abnormalities, specifically trisomies 2, 7, and/or 8, are associated with a more favorable course of disease when compared with patients with structural abnormalities, specifically del(1p), del(1q), del(6q), +der(18), or del(22q), or gain of an X chromosome or chromosome12, which are associated with an unfavorable outcome [29, 329]. Progression of FL to DLBCL occurs in 60–80% of cases and is accompanied by accumulation of secondary abnormalities, including +7, del(10q), del(6q), and/or +der(18) [330] (Fig. 15.16b).

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Jun 17, 2017 | Posted by in BIOCHEMISTRY | Comments Off on The Cytogenetics of Hematologic Neoplasms
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