FISH panels for hematologic malignancies

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Cytogenet Genome Res 118:284–296 (2007) DOI: 10.1159/000108312

FISH panels for hematologic malignancies

C. Sreekantaiah Clinical Cytogenetics, Dianon Systems, Stratford, CT (USA)

togenetic analysis is hampered by the low in vitro mitotic activity of leukemic cells, poor chromosome morphology, considerable karyotypic variability and complexity, or a normal karyotype, FISH analysis has provided a rapid and reliable method for determination of specific abnormalities in both mitotic and interphase cells and, because greater numbers of cells are analyzed, provided a more representa-tive assessment of the proportion of abnormal cells. FISH panels have therefore been designed to detect specific chro-mosomal abnormalities in each disease to aid in diagnosis, as well as detect those of proven clinical significance to serve as indicators of poor prognostic outcome and deter-mine the best therapeutic approach. On follow-up samples, FISH panels are useful in monitoring patients on subse-quent visits to assess the response to therapy and determine if clonal evolution and disease progression have occurred. Most laboratories use a commercially available set of DNA probes to target specific sequences on specific chromo-somes. These may be centromere-specific probes, unique

Abstract. Cytogenetic analysis of hematological malig-nancies has played a crucial role in the diagnosis and clini-cal management of patients, as well as in providing funda-mental insights into the genetic basis of the pathogenesis of these diseases. Leukemias and lymphomas have lent them-selves readily to karyotypic analysis and undoubtedly rep-resent the greatest successes of cytogenetics in human can-cer. Several cytogenetic changes have been shown to have considerable prognostic significance also and are being used as measurable targets for response to therapy. Molecu-lar characterization of the recurrent cytogenetic abnormal-ities has identified genes involved in leukemogenesis and formed a basis for specific treatment strategies. Fluores-cence in situ hybridization (FISH) analysis, since its intro-duction, has revolutionized the field and enabled a more precise determination of the presence and frequency of ge-netic abnormalities. It is particularly indispensable where

Request reprints from Chandrika Sreekantaiah, Ph.D.Director Clinical Cytogenetics, Dianon Systems, A Labcorp Company200 Watson Boulevard, Stratford, CT 06615 (USA)telephone: +1 203 380 4176; fax: +1 203 380 4554e-mail: [email protected]

© 2007 S. Karger AG, Basel1424–8581/07/1184–0284$23.50/0

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metaphase cytogenetics may be difficult in the largely qui-escent cells of some hematological malignancies, particu-larly the lymphoid disorders. FISH probes have been used extensively to detect nonrandom abnormalities in inter-phase nuclei and the true incidence of chromosome abnor-malities has been proven to be much higher than that de-tected by conventional chromosomal analysis. The avail-ability of a comprehensive line of commercial probes for rapid identification of critical genetic aberrations has con-tributed to the widespread use of this technique. It has also led to the current practice in most laboratories to test for genetic aberrations by using FISH panels that have been de-signed to detect genetic changes important not only in the diagnosis of leukemias and lymphomas, but also because of their association with prognosis, to identify high-risk popu-lations in specific hematological cancers, so they can be tar-geted for aggressive therapy. Copyright © 2007 S. Karger AG, Basel

Cytogenetic and FISH analyses are currently integral components in the diagnosis and management of patients with hematological malignancies. Whereas cytogenetic analysis of these disorders led to the identification of dis-tinctive chromosomal abnormalities that are often unique-ly associated with morphologically and clinically distinct subsets of leukemias and lymphomas, FISH analysis in cer-tain cancers, particularly the lymphoid malignancies, has resulted in the expansion of these capabilities and become an invaluable adjunct by identifying the specific genetic changes in non-dividing interphase cells. In cases where cy-

Manuscript received 1 June 2007; accepted in revised form for publication by L. Cannizzaro, 18 June 2007.

http://dx.doi.org/10.1159%2F000108312

Cytogenet Genome Res 118:284–296 (2007) 285

sequence probes, fusion probes or break-apart probes. The probes are hybridized either singly or more commonly in multi-color combinations allowing simultaneous detection of multiple chromosomes and/or chromosome regions. The number of interphase nuclei scored range from 100 to 500 per probe in different laboratories and, when the percentage of cells with an abnormality exceeds a cut-off value estab-lished for each probe following internal validation, is re-ported as a clonal abnormality.

The incidence of relevant cytogenetic abnormalities us-ing specifically designed probe sets in chronic lymphocytic leukemia (CLL), multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and their clinical relevance including prognostic implications is reviewed.

Chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is the most com-mon adult leukemia in the United States and Europe ac-counting for approximately 30% of all leukemia (Ries et al., 2006). The majority of patients are over 50 years old with a median age of 65 and an excess of males. The disease is char-acterized by the clonal proliferation and accumulation of neoplastic B lymphocytes in the blood, bone marrow, lymph nodes and spleen (Jaffe et al., 2001). The immunophenotype of this monoclonal population includes the pathognomic coexpression of CD5 and CD19, as well as positivity for CD20, CD21, CD23, and CD24. CLL may also present as a lymph node-based disease, called small lymphocytic lym-phoma (SLL). The clinical course is heterogeneous with sur-vival times ranging from within a few months of diagnosis, despite aggressive therapy, to many years without therapy. Clinical staging systems devised to classify patients based on the extent of the disease at initial diagnosis and provide the best predictors of survival (Rai et al., 1975; Binet et al., 1981), predicted survival duration for patients with the most advanced stage as 1–2 years, and a median survival time of over 10 years for patients with the lowest stage of disease. The exception to this system includes patients with early stage CLL. More recently, immunophenotype and genetic changes have also been shown to contribute prognostic in-formation. CD38 positivity is associated with significantly shorter overall survival and progression-free survival times (Jelinek et al., 2001; Ghia et al., 2003). Absence of hypermu-tation of the immunoglobulin heavy chain variable region (IGHV) genes present in about 50% of CLL cases is also as-sociated with a high risk for early progression (Hamblin et al., 1999; Krober et al., 2002). Expression of the intracellular signaling molecule ZAP70 has been identified in a majority of CLL cases without IGHV mutations, and is highly predic-tive of patients with more rapid disease progression and death (Crespo et al., 2003; Wiestner et al., 2003). Chromo-somal abnormalities provide independent prognostic infor-mation in newly diagnosed CLL patients. The presence of clonal aberrations, specific chromosomal abnormalities, the percentage of abnormal cells and the complexity of the

aberrations are all indicators of disease progression and survival, and have resulted in the delineation of prognostic subgroups in CLL with implications for the design of risk-adapted therapeutic strategies (Juliusson and Gahrton, 1990; Juliusson et al., 1990; Dohner et al., 2000; Thornton et al., 2004).

Conventional cytogenetic analysis of CLL has been ham-pered by low spontaneous mitotic activity and the lack of growth of leukemic cells in culture, despite appropriate stimulation with polyclonal B-cell mitogens. The incidence of clonal cytogenetic abnormalities in reported studies ranges from 28.5% (Reddy, 2006) to over 80% (Dicker et al., 2006; Schoch et al., 2006) with an average of 40–50% of cases (Juliusson and Gahrton, 1990; Escudier et al., 1993; Hernandez et al., 1995; Finn et al., 1996; Bigoni et al., 1997; Athanasiadou et al., 2006). About 10% of these have com-plex karyotypic changes. The most common cytogenetic ab-normalities account for more than 60% of all abnormal cas-es and include trisomy of chromosome 12, translocation or deletion of 13q, rearrangements involving 14q32, and dele-tions of 6q, 11q and 17p. Less frequent are trisomies of chro-mosomes 3 and 18. Because of its greater sensitivity, FISH analysis was often used initially as an adjunct to cytogenet-ic analysis in the detection of specific abnormalities in cas-es with a normal karyotype or those with insufficient or no metaphases. These analyses showed not only an increase in the detection rate of abnormalities to about 80% (Dohner et al., 1999, 2000; Dewald et al., 2003), but also a difference in the spectrum of chromosomal abnormalities detected. Ab-normalities that were cytogenetically rare were observed in greater frequency by FISH, such as deletion of 13q, 11q and 17p, and those that were common by routine chromosome analysis were observed to be not as frequent, e.g. trisomy 12.

FISH panels were therefore designed to reliably detect genetic abnormalities of proven clinical significance. The DNA probe set generally includes probes for chromosome 12 centromere for detection of trisomy 12, ATM (11q22.3), D13S25 (13q14.3), D13S319 (13q14.3), RB1 (13q14), LAMP1 (13q34) and TP53 (17p13.1) for deletions of 11q, 13q and 17p, respectively, and IGH (14q32.3) for rearrangements at that locus. MYB probe to determine deletion of 6q has also been included by some laboratories.

Deletion 13q Deletion of the long arm of chromosome 13 was reported

in 10–20% of cases by cytogenetics (Juliusson et al., 1990; Escudier et al., 1993; Athanasiadou et al., 2006). By FISH analysis it is the most common abnormality and is present in about 31–55% of cases (Dohner et al., 2000; Dewald et al., 2003; Aoun et al., 2004; Goorha et al., 2004; Gozzetti et al., 2004; Glassman and Hayes, 2005; Sindelarova et al., 2005; Reddy, 2006). The deletion is seen as the sole abnormality or in association with other aberrations, and in either the hemizygous state (60–70%), or the homozygous and mosaic states in equal proportions (10–20% each) (Dohner et al., 2000; Dewald et al., 2003; Aoun et al., 2004; Fink et al., 2004; Reddy, 2006). Prognostically, patients with deletion 13q

Cytogenet Genome Res 118:284–296 (2007)286

have the overall best disease free survival times, akin to those with normal karyotypes (Juliusson and Gahrton, 1990; Dohner et al., 2000). However, even within this sub-group of CLL the clinical course of the disease may be het-erogeneous. Patients with a homozygous deletion fare worse than the hemizygous cases. Also, patients with deletion 13q and other abnormalities have an unfavorable prognosis (Dewald et al., 2003; Fink et al., 2004).

Trisomy 12 Trisomy 12 was the most common abnormality observed

cytogenetically with an incidence of 8–20% of all cases (Ju-liusson et al., 1990; Escudier et al., 1993; Que et al., 1993; Athanasiadou et al., 2006), but by FISH it is the second most common abnormality and has been observed in 7–35% of the cases (Anastasi et al., 1992; Cuneo et al., 1992; Escudier et al., 1993; Que et al., 1993; Matutes et al., 1996; Aoun et al., 2004; Goorha et al., 2004; Gozzetti et al., 2004; Glassman and Hayes, 2005; Sindelarova et al., 2005; Reddy, 2006). Tri-somy 12 is strongly associated with atypical lymphocyte morphology, atypical marker expression, advanced stage of the disease and resistance to chemotherapy (Cuneo et al., 1992; Coignet et al., 1993; Escudier et al., 1993; Que et al., 1993). Expression of FMC7, CD38, CD20 and surface im-munoglobulin light chain was significantly higher (Finn et al., 1996; Matutes et al., 1996; Ghia et al., 2003; Athanasia-dou et al., 2006; Reddy, 2006). One study found that patients with trisomy 12 tended to present with more splenomegaly than those without and this was consistent with the more advanced stage seen in trisomy 12 patients (Escudier et al., 1993). They were also more likely to be previously treated. Prognostically, trisomy 12 patients had significantly shorter median survival times compared to those with deletion of 13q as the sole aberration (Dohner et al., 2000) and those with normal karyotypes in some studies (Juliusson et al., 1990; Escudier et al., 1993; Criel et al., 1997), while other analyses found no significant difference in survival between patients with trisomy 12 and normal karyotypes (Athanasi-adou et al., 2006). Based on studies combining FISH and immunophenotyping and the observation of trisomy 12 in low numbers of trisomic cells in patients with other abnor-malities, it has been suggested to be a secondary event in leukemogenesis in some patients (Escudier et al., 1993).

Deletion 11q Deletion of 11q was not identified as a frequent aberra-

tion by routine chromosome analysis, but by FISH analysis it was determined to be the third most frequent abnormal-ity, observed in up to 23% of the cases (Dohner et al., 1997, 2000; Neilson et al., 1997; Dewald et al., 2003; Aoun et al., 2004; Goorha et al., 2004; Gozzetti et al., 2004; Glassman and Hayes, 2005; Sindelarova et al., 2005; Reddy, 2006). Clinically, patients with deletion 11q were younger, had more advanced clinical stages and were characterized by extensive peripheral, abdominal and mediastinal lymph-adenopathy. The younger patients (age group less than 55 years) showed a more rapid disease progression with short-er survival times (Dohner et al., 1997; Neilson et al., 1997).

This was independent of the stage of disease and may there-fore represent an early marker for aggressive disease.

Deletion 17p Deletions of 17p have been observed in 7–15% of patients

by FISH and correlate with advanced disease, resistance to treatment and poor survival (el Rouby et al., 1993; Dohner et al., 1995, 2000; Geisler et al., 1997). Also, the development of 17p deletion is associated with mutations or deletions of the tumor suppressor gene TP53 and is more likely to occur as a result of clonal evolution after initial diagnosis.

Deletion 6q Deletions of 6q are commonly observed nonrandom

chromosomal aberrations in lymphoid malignancies. In CLL, they have been reported cytogenetically in about 4–21% of cases, with an average of 7% by FISH analysis (Amiel et al., 1999; Stilgenbauer et al., 1999; Dohner et al., 2000; Cuneo et al., 2004; Reddy, 2006). Two regions of minimal deletion have been established and include 6q21–q23 and 6q25–q27. Patients with deletion 6q had higher white blood cell counts, frequent splenomegaly, more extensive lymph-adenopathy, atypical morphology, CD38 positivity and short to intermediate survival (Bigoni et al., 1997; Stilgen-bauer et al., 1999; Cuneo et al., 2004; Reddy, 2006). This deletion is not routinely screened for, however, because of its association with poor prognosis it might be a good can-didate for inclusion in CLL FISH panels.

Rearrangements of 14q32 Earlier studies of chromosomal abnormalities in CLL

found 14q32 rearrangements in a high percentage of cases and an association with poorer survival compared to abnor-malities of 13q (Juliusson et al., 1990). However, with the current reclassification of t(11; 14) with CCND1/IGH rear-rangement as mantle cell lymphoma as well as other lym-phoma associated translocations, fewer cases of CLL, about 4–5%, have abnormalities of 14q32 (Dohner et al., 2000; Aoun et al., 2004; Mayr et al., 2006). Patients with a rear-rangement involving 14q32 have an unfavorable progno-sis.

Multiple myeloma (MM)

Multiple myeloma (MM) is a heterogeneous mature B-cell lymphoid disorder characterized by a serum mono-clon al protein and skeletal destruction with osteolytic le-sions, pathological fractures, bone pain, hypercalcemia and anemia (Jaffe et al., 2001). It is the most common lymphoid malignancy in Blacks and the second most common in Whites, representing 15% of all hematological malignan-cies. The median age of diagnosis is 68 years in males and 70 years in females. The male to female ratio is approxi-mately 1: 1 (Jaffe et al., 2001). The clinical course of MM pa-tients is highly variable and survival times range from a few months to several years (Kyle et al., 2003). Prognostic factors include clinical staging (Durie and Salmon, 1975), plasma-


cell proliferation (Greipp et al., 1993), plasma-cell morphol-ogy (Fritz et al., 1984), serum levels of beta-2 microglobulin, C-reactive protein (CRP) and lactate dehydrogenase (LDH) (Dimopoulos et al., 1991; Bataille et al., 1992). Advanced stages of the disease, plasmablastic morphology, � 2 micro-globulin, and high serum CRP and LDH levels are associ-ated with a poor prognosis (Rajkumar and Greipp, 1999). More recently, cytogenetics has emerged as another impor-tant independent prognostic indicator helping to define a high-risk population that would benefit from intensive therapy. Abnormal karyotypes were consistently associated with a rapidly fatal outcome, and fewer than 10% of patients with these abnormalities survived longer than five years. Konigsberg et al. (2000) found that in the presence of ad-verse cytogenetic aberrations other prognostic factors of clinical importance in univariate analysis no longer have independent significance. In particular, deletions of 13q and 17p, and 11q rearrangements, as well as cases with a t(11; 14), were determined to be unfavorable cytogenetic abnor-malities (Drach et al., 1998; Fonseca et al., 1999a; Konigs-berg et al., 2000; Zojer et al., 2000; Facon et al., 2001; Chang et al., 2005).

Extensive cytogenetic analyses have been carried out on MM patients, but the generally low mitotic activity resulted in an underestimation of the true incidence of abnormali-ties. In most published series, cases with abnormal karyo-types ranged from approximately 30 to 50% (Dewald et al., 1985; Weh et al., 1993; Lai et al., 1995a; Sawyer et al., 1995; Debes-Marun et al., 2003; Pantou et al., 2005). Complex karyotypes with multiple chromosomal abnormalities are the rule. Most MM cases are aneuploid and recurrent nu-merical aberrations include trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21, and losses of the sex chromosomes, 8, 13, 14, 16 and 22. Of the structural abnormalities, the most consistent ones include translocations involving 14q32 with various chromosome partners, most commonly 4, 11, 16 and 6. Rearrangements of chromosome 1 are also frequent; however, the aberrations are diverse involving both the short and long arms, and their variable nature suggests they are secondary events. Deletion of the long arm of chromo-some 13 is also consistently noted and together with mono-somy of chromosome 13 suggests that total or partial loss of chromosome 13 is a recurrent event. Other recurrent abnor-malities include deletions of 11q, 17p13 and 19q13.

With the use of FISH, identification of specific deletions and rearrangements, not possible by conventional cytoge-netics, raised the percentage of cases with abnormalities to up to 90% (Drach et al., 1995; Zandecki et al., 1996; Schmidt-Wolf et al., 2006; Avet-Loiseau et al., 2007), particularly when analysis was performed on purified bone marrow plasma cells. It also became clear that the delineation of spe-cific cytogenetic abnormalities had clinical and biological implications and MM patients could be divided into sub-groups based on their karyotypic profile (Tricot et al., 1995, 1997; Fonseca et al., 1999b, 2002a, b; Konigsberg et al., 2000; Zojer et al., 2000; Facon et al., 2001; Avet-Loiseau et al., 2002; Moreau et al., 2002; Dewald et al., 2005). The most frequent abnormalities detected by FISH analyses were de-

letion of the RB1 gene on 13q14, translocations involving the heavy chain gene on 14q32, most commonly t(11; 14), t(4; 14) and t(14; 16), gain of 11q and deletion of 17p. Because of the increased detection rate of genetic abnormalities by FISH and its immense prognostic value, the current practice in cytogenetic laboratories is to test for genetic abnormalities in MM and its precursor condition, monoclonal gammopa-thy of undetermined significance (MGUS) by FISH. The FISH panel for MM in most laboratories has been designed to include probes for the detection of deletions of 11q ( MLL at 11q23), 13q ( RB1 at 13q14 and LAMP1 at 13q34) and 17p (TP53) , and rearrangement of the IGH gene at 14q32 par-ticularly with CCND1 at 11q13. Some laboratories also in-clude probes for 4p16.3 (FGFR3) and 16q23 (MAF) for detec-tion of translocations with the IGH gene. In addition to this, studies have recommended inclusion of probes to deter-mine gains of regions 1q, 9q and 11q (Liebisch et al., 2003).

The most frequent cytogenetic abnormalities and their clinical attributes are discussed in detail.

Aneuploidy Aneuploidy is a common characteristic of MM (Dewald

et al., 1985; Drach et al., 1995; Lai et al., 1995a; Sawyer et al., 1995; Zandecki et al., 1996; Debes-Marun et al., 2003) and is independent of clinical stage. Karyotypes may be hypo-diploid, hyperdiploid, or pseudodiploid with balanced structural rearrangements. Ploidy category has been shown to have a significant effect on prognosis. Patients with a nor-mal karyotype have a better prognosis than those with chromosomal abnormalities. Hypodiploidy has been asso-ciated with a poor prognosis (Smadja et al., 2001; Fassas et al., 2002; Debes-Marun et al., 2003). This, however, may not be independent of other adverse cytogenetic abnormalities such as 13q deletions and 14q translocations that are pre-dominant in hypodiploid MMs (Fonseca et al., 2003). Hy-perdiploidy was seen in 39% of patients and was observed to have marginal prognostic impact (Avet-Loiseau et al., 2007).

Deletion 13q Deletion of 13q or monosomy of chromosome 13 was re-

ported in 10–20% of MM cases by routine cytogenetic anal-ysis (Weh et al.,1993; Lai et al., 1995a; Sawyer et al., 1995; Debes-Marun et al., 2003). By FISH analysis partial deletion of 13q or loss of chromosome 13 is one of the most common abnormalities in MM and has been reported in 30–50% of patients (Zojer et al., 2000; Facon et al., 2001; Fonseca et al., 2002b, 2003; Chang et al., 2004a; Avet-Loiseau et al., 2007). About 85% of deletion 13q cases detected by interphase FISH analysis represent monosomy 13 and only 15% are in-terstitial deletions (Avet-Loiseau et al., 2000; Fonseca et al., 2001). The minimal area of deletion, although not precisely mapped, appears to predominantly involve the RB1 gene and the D13S319 locus at 13q14.3 (Shaughnessy et al., 2000; Zojer et al., 2000). The deletions are mostly monoallelic. Deletion of 13q/monosomy 13 is an independent prognostic variable on multivariate analysis (Zojer et al., 2000; Facon et al., 2001; Fonseca et al., 2002b). It is associated with short-


er survival, a lower likelihood of response to therapy and advanced stages of the disease (Desikan et al., 2000; Zojer et al., 2000; Facon et al., 2001; Fonseca et al., 2002b; Shaugh-nessy et al., 2003). A recent study, however, determined that the prognostic power of deletion 13q was related to t(4; 14) and deletion of 17p which are often associated with deletion of 13q (Avet-Loiseau et al., 2007). Clinically, 13q loss is also associated with lambda-light chain type, low serum mono-clonal concentration of ̂ 1 q/dl and higher PC labeling in-dex (Facon et al., 2001; Fonseca et al., 2002b).

14q32 translocations Cytogenetic analysis revealed that 14q32 was one of the

most frequent chromosomal breakpoints. Rearrangements occurred at this band in about 10–60% of patients (Lai et al., 1995a; Sawyer et al., 1995; Pantou et al., 2005) with various chromosomal partners, most commonly chromosomes 11 and 8. FISH analysis revealed that the translocations involve the immunoglobulin heavy chain (IGH) locus at 14q32, are mostly cryptic and present in 40–75% of patients. The most common partners are 11, 4, 16 and 6 (Bergsagel et al., 1996; Nishida et al., 1997; Fonseca et al., 1999a; Avet-Loiseau et al., 1999, 2002; Chang et al., 2004a; Schmidt-Wolf et al., 2006), although other chromosomal regions may be involved. All the translocations lead to upregulation of the partner gene. The translocations represent a significant prognostic factor in MM with the rate of 14q32 rearrangements increasing with disease progression and reaching 90% in advanced cancers. These translocations are also present in MGUS and are therefore considered to be early pathogenetic events (Fonseca et al., 2002c). In MGUS, however, the transloca-tions have no effect on prognosis.

t(11; 14)(q13;q32) This translocation has been identified in 15–20% of pa-

tients with MM (Avet-Loiseau et al., 2002, 2007; Fonseca et al., 2002a, 2003; Gertz et al., 2005) and 15–30% of patients with MGUS (Avet-Loiseau et al., 2002; Fonseca et al., 2002a). The t(11; 14) correlates with a lymphoplasmacytic, mature morphology of plasma cells, CD20 expression and the oli- go-/asecretory MM subtype (Hoyer et al., 2000; Fonseca et al., 2002a; Moreau et al., 2002; Avet-Loiseau et al., 2003; Robillard et al., 2003). The translocation results in overex-pression of cyclin D1 akin to the situation in mantle cell lymphoma. The t(11; 14) has been associated with good prognosis in MM patients receiving high-dose chemother-apy and stem cell transplant (Moreau et al., 2002; Soverini et al., 2003). Avet-Loiseau et al. (2007), however, reported that it does not influence prognosis.

t(4; 14)(p16.3;q32) This cryptic translocation present in about 13–20% of

cases and detectable by FISH, leads to the upregulation of the oncogenes Multiple Myeloma SET (MMSET or WHSC1) on 4p and FGFR3 on 14q, respectively (Chesi et al., 1998; Fonseca et al., 2001, 2003; Avet-Loiseau et al., 2007). Patients with the translocation commonly have an IgA isotype, ag-gressive clinical features and are associated with poor prog-

nosis after high dose therapy (Moreau et al., 2002; Fonseca et al., 2003; Keats et al., 2003; Chang et al., 2004b; Gertzet al., 2005; Avet-Loiseau et al., 2007). When evaluated to-gether with � 2 microglobulin levels, however, patients with t(4; 14) and a low � 2 microglobulin had longer survival, close to that of patients without the translocation but with a high � 2 microglobulin level (Avet-Loiseau et al., 2007). A strong correlation between t(4; 14) and deletion 13q was noted and appeared to influence the prognostic impact of deletion 13q conferring an especially aggressive phenotype (Fonseca et al., 2001, 2003; Moreau et al., 2002).

t(14; 16)(q32;q23) This translocation is also more reliably detected by FISH

and is present in about 2–10% of cases (Chesi et al., 1998; Avet-Loiseau et al., 2002; Fonseca et al., 2003). It results in the upregulation of the transcription factor c-MAF and cyclin D2. The prognostic correlation is not clear; how-ever, Fonseca et al. (2003) noted a shorter survival and fea-tures of aggressiveness in patients with the translocation. A significant positive correlation appears to exist betweent(14; 16) and deletion of 17p13.1 (Fonseca et al., 2003).

t(6; 14) This translocation is relatively rare and has been ob-

served in 3–4% of MM patients (Sawyer et al., 2001). It is associated with the upregulation of cyclin D3 (Shaughnessy et al., 2001). There is no known clinical or prognostic infor-mation for this translocation yet.

Deletion 17p Deletions of the TP53 tumor suppressor gene at 17p13.1,

as detected by FISH analysis, are predominantly monoal-lelic and range from about 5 to 33% in newly diagnosed MM patients (Drach et al., 1998; Fonseca et al., 2003; Chang et al., 2005; Avet-Loiseau et al., 2007). In patients with relapsed MM the incidence was about 55% (Drach et al., 1998). Loss of 17p occurs either due to an unbalanced rearrangement, an interstitial deletion or a translocation involving 17p, or monosomy of chromosome 17. Deletion of TP53 appears to be a marker of disease progression, functional loss of the gene by deletion or mutation was present in about 40% of patients with advanced MM (Neri et al., 1993). Deletion of 17p is associated with significantly worse prognosis (Drach et al., 1998; Fonseca et al., 2003; Chang et al., 2005; Gertz et al., 2005) and the patients also have other features of aggres-siveness such as plasmacytoma and hypercalcemia (Fonseca et al., 2003).

Other abnormalities Chromosome 1 rearrangements . Structural abnormali-

ties of chromosome 1 involve both the short arm and the long arm and have been observed cytogenetically in 15–50% of patients (Lai et al., 1995a; Sawyer et al., 1995; Segeren et al., 2003). A strong association between chromosome 1 ab-normalities and those of chromosome 13 was also noted (Segeren et al., 2003). Chromosome 1 abnormalities ap-peared to define a high-risk population with advanced dis-


ease, shorter event-free survival, time to progression and overall survival (Sawyer et al., 1995; Segeren et al., 2003; Bang et al., 2006). Amplification and overexpression of the cell cycle regulator gene CKS1B at 1q21 was shown to predict an aggressive clinical course (Shaughnessy, 2005) and this is consistent with the adverse prognostic features observed by Bang et al. (2006) in their cases with trisomy 1q.

9q rearrangements . Gain of 9q is also associated with very poor prognosis, particularly in patients with deletion of 13q also (Liebisch et al., 2005).

Trisomy 11. Trisomy of chromosome 11 has been report-ed in 25–66% of cases (Harrison et al., 2003; Cremer et al., 2005; Bang et al., 2006; Guglielmelli et al., 2007). The prog-nostic impact of this gain is disputed; while Konigsberg et al. (2000) determined that it had no prognostic value, Gu-tierrez et al. (2004) reported shorter survival in patients with gains of 11q.

Deletion of 6q27. Deletion of the long arm of chromo-some 6 is associated with various lymphoproliferative dis-orders. In MM loss of 6q, particularly band 6q21, was one of the most frequent losses and it was detected in 10–28% of patients (Cigudosa et al., 1998; Amiel et al., 1999; Gutierrez et al., 2004).

Coexistence of abnormalities. Patients with more than one abnormality often have a worse prognosis. Fonseca et al. (2003) observed a single chromosome abnormality in 40%, two abnormalities in 22% and three abnormalities in 3% of MM patients. Certain abnormalities are found in association with many other abnormalities, for instance de-letion of 13q has been reported in 36% of hyperdiploidpatients, in 39% with t(11; 14), 85% with t(4; 14), 92% with t(14; 16) and 78% with deletion 17p. Loss of 17p occurred with t(14; 16) in 33% of patients, with t(4; 14) in 20% and a low incidence was seen with t(11; 14). Few patients were seen where t(4; 14) and t(11; 14) occurred together with hyperdip-loidy. No association of t(4; 14) and t(11; 14) has been report-ed (Avet-Loiseau et al., 2002, 2007; Dewald, 2005). Other genetic mutations have been identified in MM and involve RAS, MYC, TP53, PTEN and various components of the RB pathway (Fonseca et al., 2004). All of these factors also have an impact on survival (Avet-Loiseau et al., 2002, 2007; Fon-seca et al., 2003; Dewald, 2005).

Acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous clon-al disease of hematologic progenitor cells. Its salient feature is the excessive accumulation of myeloid blasts in bone mar-row, peripheral blood and other tissues. It is the most com-mon malignant myeloid disease in adults with a median age at presentation of 70 years and a male to female ratio of 3: 2. Etiologic factors include viruses and exposure to ionizing radiation, benzene and cytotoxic chemotherapy (Jaffe et al., 2001; Estey and Dohner, 2006). Up to 10–15% of patients with AML are therapy-related and develop the disorder af-ter treatment with cytotoxic agents, some 5–10 years after exposure to alkylating agents and others 1–5 years after

treatment with topoisomerase II inhibitor drugs such as doxorubicin and etoposide (Estey and Dohner, 2006). Each of these types is associated with specific chromosomal aber-rations. Prognostic factors in AML include age, immuno-phenotype, leukocyte count, previous history of MDS, and levels of LDH, serum albumin, bilirubin and creatinine (Chang et al., 2004c; Gupta et al., 2005); however, cytoge-netic and molecular genetic findings at diagnosis constitute one of the most important prognostic determinants (Grim-wade et al., 1998; Mrozek et al., 2000; Slovak et al., 2000; Schoch et al., 2001; Byrd et al., 2002).

Cytogenetic abnormalities are present in an average of 55% (range 50–80%) of adults at diagnosis (Stasi et al., 1993; Heim and Mitelman, 1995; Mrozek et al., 2001; Estey and Dohner, 2006). A great majority of them are recurrent and in addition to being a critical component of the armamen-tarium of the diagnostic workup of AML, cytogenetic char-acterization is a basis for formulating therapeutic strategies (Tallman et al., 1997; Bloomfield et al., 1998). It is also a critical independent predictive factor of prognosis, response to chemotherapy, risk of relapse and outcome (Grimwade et al., 1998). Some of the abnormalities are associated with re-markable specificity with morphological and cytochemical hematologic subtypes as defined by the FAB classification (Bennett et al., 1976; Heim and Mitelman, 1995) and cur-rently the WHO classification incorporates cytogenetics and molecular genetics in an attempt to define entities that are biologically homogeneous and prognostically relevant (Vardiman et al., 2002). Four abnormalities, t(15; 17), t(8; 21), inv(16) and t(16; 16), are seen in nearly 30% of patients with AML (Grimwade et al., 1998; Mrozek et al., 2000; Slovak et al., 2000) and have a strong correlation with morphology, as well as distinctive clinical findings and favorable response to therapy. These are included in the subgroup AML with recurrent genetic abnormalities, and emerging new infor-mation may result in the expansion of this subgroup in the future. The other major WHO categories include AML with multilineage dysplasia, therapy-related AML and MDS, and AML not otherwise categorized. The therapy-related AMLs and MDSs are also characterized by specific chromosomal abnormalities. Alkylating agent/radiation-related t-AML and t-MDS show a higher incidence of abnormalities of chromosomes 5 and 7 and have a worse clinical outcome. Topoisomerase II inhibitor-related AML does not have a preceding MDS phase and is associated with balanced translocations involving 11q23 or 21q22, as well as inv(16) and t(15; 17) (Vardiman et al., 2002).

Numerous other recurrent structural and numerical ab-normalities are present in AML (Heim and Mitelman, 1995; Mrozek et al., 2001) and some of the more common ones include monosomy or deletion of 5/5q, monosomy or dele-tion of 7/7q, translocations involving 11q23 with multiple chromosome partners, t(6; 9), deletions of 11q and 20q, rear-rangements of 3q36, and trisomies of chromosomes 8, 9, 11, 13, 19, 21 and 22. Secondary aberrations often accompany the primary change and can cause substantial variability in a patient’s outcome. Molecular characterization of the spe-cific rearrangements has implicated genes important in cel-


lular proliferation and differentiation (Cline, 1994) and demonstrated the creation of fusion genes that encode chi-meric proteins responsible for leukemogenesis.

The AML FISH panel is extensive and is used to detect known losses and gains, or specific translocations and rear-rangements of chromosomes. The probes commonly used are EGR1 at 5q31 and D5S23, D5S721 at 5p15.2 for detection of monosomy/deletion of 5/5q, CEP7 and D7S486 for loss/deletion of 7/7q, CEP8 for gain of chromosome 8, ETO/AML1 ( RUNX1T1/RUN X1, 8q22/21q22) for the t(8; 21) rear-rangement, RARA at 17q21 for the t(15; 17) (PML/RARA) rearrangement, CBFB at 16q22 for the inv(16) rearrange-ment, and MLL for rearrangements of 11q.

Prognostically, based on cytogenetic and molecular ge-netic findings at diagnosis, AML patients can be broadly divided into those with favorable, intermediate or adverse outcomes (Grimwade et al., 1998, 2001; Slovak et al., 2000; Byrd et al., 2002). Within each group, particularly the favor-able and intermediate groups, there is variability in out-come depending on secondary chromosomal abnormali-ties, gene mutations and deregulated gene expression (Estey and Dohner, 2006).

Prognostically favorable group This group is characterized by low rates of primary drug

resistance and superior overall survival associated with a reduced recurrence risk. Additional cytogenetic aberra-tions in this group did not in general have a deleterious ef-fect on outcome (Grimwade et al., 1998).

t(8; 21). This translocation is present in approximately 30% of AML with maturation. The AML1 (RUNX1) gene on 21q22 is juxtaposed with the ETO (RUNX1T1) gene on 8q22 resulting in a novel chimeric gene AML1/ETO (RUNX1/RUNX1T1) . Loss of the Y chromosome, when present in as-sociation with t(8; 21), is associated with shorter survival in men (Schlenk et al., 2004). Also, deletion 9q as an addition-al change has been reported as a poor risk indicator requir-ing more aggressive treatment (Schoch et al., 1996).

t(15; 17) . This translocation is characteristic of acute pro-myelocytic leukemia (APL) in which abnormal promyelo-cytes predominate. The retinoic acid receptor alpha (RARA) gene on 17q12 fuses with the PML gene on 15q22 to produce a PML/RARA gene fusion product. The translocation is de-tectable by FISH with the PML-RARA probe set. A RARA break-apart probe can also be used to detect t(15; 17), as well as variant translocations associated with APL involving the RARA gene with genes other than PML . Patients with t(15; 17) are sensitive to treatment with all trans retinoic acid with significant improvement in outcome (Fenaux et al., 1997; Tallman et al., 1997).

inv(16), t(16; 16) . These rearrangements are seen in 10–12% of AML. Both rearrangements result in the fusion of the CBFB gene at 16q22 with the MYH11 gene at 16p13 giv-ing rise to a chimeric protein. The abnormality is detectable by FISH using a CBFB break-apart probe. Trisomy of chro-mosome 22, when present, improves relapse-free survival (Schlenk et al., 2004; Marcucci et al., 2005).

Intermediate prognosis group This subgroup is very heterogeneous and patients with a

normal karyotype constitute the largest proportion of pa-tients ( � 88%). The remainder includes gains of chromo-somes 6, 8, 11, 13, 21 and 22, loss of the Y chromosome, de-letions of 7q, 9q, 12p and 20q, and abnormalities other than those noted in the other two categories (Grimwade et al., 1998; Slovak et al., 2000; Estey and Dohner, 2006). Their complete remission rate, risk of relapse and outcome are worse than those of treated patients in the prognostically favorable group described above but better than that of pa-tients belonging to the adverse group with unfavorable cy-togenetic findings. Additional chromosome abnormalities in this group, however, had a deleterious effect on outcome. Patients with a normal karyotype represent about 45% of patients with AML. At the molecular level, however, these patients are very heterogeneous and contain several prog-nostically significant gene mutations and changes in gene expression (Estey and Dohner, 2006; Mrozek et al., 2007).

Group with a poor prognosis Patients in this group had a significantly poorer outcome

than patients with a normal karyotype. They had resistant disease and were less likely to achieve complete remission and had a poorer overall survival reflecting increased risk of death on induction and/or relapse. Additional chromo-somal abnormalities in this group did not affect the out-come. This group included patients with a complex karyo-type, i.e. three or more abnormalities, monosomies of chro-mosomes 5 and 7, deletion of 5q, abnormalities of 3q (inv(3), t(3; 3)), 11q (t(9; 11) and t(11; 19)), 20q, 21q, deletion of 9q, t(9; 9) and t(9; 22) (Grimwade et al., 1998, 2001; Slovak et al., 2000; Schoch et al., 2001; Byrd et al., 2002; Estey and Doh-ner, 2006; Chen et al., 2007). A few rearrangements, t(6; 9), deletion 7q and 11q23 rearrangements, were classified as unfavorable by some groups (Buchner et al., 1999; Slovak et al., 2000; Byrd et al., 2002) and intermediate by others (Grimwade et al., 1998) reflecting in part the heterogeneity of the 11q23 abnormalities.

Myelodysplastic syndrome (MDS)

The myelodysplastic syndromes (MDS) are a clinically heterogeneous group of hematologic diseases occurring predominantly in older adults (median age 76 years) with a significantly higher incidence in men (Ma et al., 2007). They are characterized by bone marrow dysplasia in one or more myeloid cell lineages, peripheral blood cytopenia, most commonly anemia and less frequently neutropenia and/or thrombocytopenia, and frequent progression to acute my-eloid leukemia (AML). The number of myeloblasts in the blood or bone marrow is less than the 20% requisite for a diagnosis of AML (Vardiman et al., 2002). MDS may occur de novo or as a result of therapy with either alkylating agents or radiotherapy. They have an overall short survival, death being generally due to the consequences of cytopenia or progression to AML (Jaffe et al., 2001). Survival has been


correlated with age at diagnosis, gender, clinical subtype, bone marrow morphology and the percentage of bone mar-row blast cells. Cytogenetic analysis is an accepted indepen-dent predictor of clinical outcome and overall survival, and of the likelihood of progression to AML. Several studies have demonstrated its value in determining prognosis and as a basis for targeted therapies (Aul et al., 1992; Morel et al., 1993; Toyama et al., 1993; Greenberg et al., 1997; Nevill et al., 1998; Pfeilstocker et al., 1999; Sole et al., 2000; Malco-vati et al., 2005). An International MDS Risk Analysis Workshop classified untreated primary MDS into cytoge-netic subgroups with good, intermediate and poor progno-ses and these were incorporated into an International Prog-nostic Scoring System (IPSS) (Greenberg et al., 1997).

Routine cytogenetic analysis has demonstrated chromo-somal aberrations in approximately 40–70% of patients with primary MDS at diagnosis and in 95% of patients with t-MDS (Heim and Mitelman, 1995; Fenaux et al., 1996; Val-lespí et al., 1998; Olney and Le Beau, 2001; Mauritzson et al., 2002). Numerous recurrent structural and numerical chro-mosomal abnormalities have also been identified in MDS and the most commonly observed are losses of chromo-somes 5 and/or 7, deletions of the long arm of chromosomes 5, 7, and/or 20, and trisomy of chromosome 8. Less frequent-ly observed abnormalities include structural abnormalities of chromosome 1, the long arm of chromosome 3, deletions of the long arms of chromosomes 11 and 13, loss of the short arm of chromosome 17 due to monosomy or an unbalanced rearrangement, and trisomy of chromosomes 9 and 21. The abnormalities may occur as a sole aberration or as part of complex karyotypes with multiple abnormalities. Many of these changes also characterize AML and the remarkable karyotypic similarity between MDS and AML emphasizes the pathobiologic similarity between the two disorders.

FISH panel testing in MDS has most commonly used probes specific for chromosomes 5 ( EGR1 at 5q31 and D5S23, D5S721 at 5p15.2), 7 (D7S486 at 7q31 and CEP7, the centromere probe), 8 (also the centromere probe), and 20 (D20S108 at 20q12), and in some reports chromosomes 11 ( MLL at 11q23) and 13 ( RB1 at 13q14) as well.

FISH analyses in MDS have for the most part shown that it is nearly as sensitive as routine cytogenetic analysis when used independently, with a slight increase in detection rate of abnormalities when both methods are used in combina-tion since some abnormalities go undetected by either ap-proach (Romeo et al., 2002). Studies favoring the use of FISH have shown that about 15–20% of MDS patients with a normal karyotype have minor clones of cells (about 15–30% of interphase nuclei) with abnormalities or submicro-scopic rearrangements that are clinically relevant and influ-ence therapy (Bernasconi et al., 2003, 2006). Such studies have also shown that FISH abnormalities were observed more frequently among patients with increased bone mar-row blasts, a higher rate of progression to AML and were predictive of worse prognosis (Rigolin et al., 2001). Others have indicated that FISH panel testing has limited utility in MDS except in cases where cytogenetic analysis is not pos-sible (Ketterling et al., 2002; Cherry et al, 2003; Sun et al.,

2004), such as in specimens with low or no mitotic cells, poor chromosome morphology, or specimens delayed in transit. They conclude that in contrast to its application in lymphoid malignancies, FISH panel testing for MDS ap-pears to not be an efficient and cost-effective screening method in the diagnosis of MDS and that routine cytoge-netic analysis should remain the method of choice for study-ing bone marrow aspirates since it provides an overall pic-ture.

Based on the cytogenetic pattern, according to the IPSS for MDS (Greenberg et al., 1997), patients were separated for both survival and AML evolution into three prognostic sub-groups, good, intermediate or poor.

Good prognosis group Patients with favorable outcomes constituted about 50–

70% of the patients and had a normal karyotype, loss of the Y chromosome, deletion 5q, or deletion 20q as the sole changes.

Normal karyotype . About 30–60% of patients with MDS have a normal karyotype. This subgroup is heterogeneous and contains abnormalities not detected by routine cytoge-netic analysis. By FISH analysis up to 18% of such cases have been found to carry common chromosomal aberrations(–5/5q–, –7/7q–, +8, +11q, 17p–) in 15–32% of cells analyzed (Rigolin et al., 2001; Bernasconi et al., 2003, 2006). These clones of abnormal cells may be clinically relevant because they identify a subset of patients within this group that may have an inferior prognosis.

Deletion 5q . Deletion of 5q has been reported in about 10–20% of de novo MDS patients and about 40% of t-MDS (Vallespí et al., 1998). Patients with deletion of 5q have a relatively good prognosis when it is present as the sole karyo-typic abnormality. However, when additional abnormalities are present the prognosis is poor with early progression to leukemia, resistance to treatment and short survival (Jacobs et al., 1986; Giagounidis et al., 2004). Transfusion-depen-dent, low or intermediate risk MDS patients with deletion 5q receive targeted therapy with lenalidomide resulting in durable cytogenetic remission and hematological responses in the majority of patients (List et al., 2006; Nimer, 2006).

Deletion 20q . Deletion of 20q is present in about 4–5% of patients with MDS and 7% of t-MDS (Fenaux et al., 1996; Vallespí et al., 1998). It is associated with low-risk disease, low rate of progression to AML, and when present as the sole abnormality, patients with deletion of 20q have a favorable prognosis (Wattel et al., 1993; Greenberg et al., 1997). How-ever, when deletion of 20q is part of a complex karyotype, the prognosis is less favorable with a high rate of transfor-mation to acute leukemia (Campbell and Garson, 1994; Brezinova et al., 2005). The most frequent additional abnor-malities were deletions of 5q and/or 7q.

Loss of the Y chromosome . Y chromosome loss has been described in hematologic malignancies as well as in bone marrows of hematologically normal elderly men (UKCCG, 1992). Thus, although this finding does not indicate the presence of MDS, when present in patients diagnosed with MDS the prognosis is improved (Greenberg et al., 1997).


Poor prognosis group About 16–32% of patients belonged to this group. Pa-

tients with abnormal cytogenetic findings carried a higher risk of progression to AML than those with a normal karyo-type and those with complex karyotypes (three or more ab-normalities) or abnormalities of chromosome 7 progressed more often to AML (Morel et al., 1993; Greenberg et al., 1997).

Complex karyotype . Complex karyotypes are variably defined and include multiple structural rearrangements (more than three), the presence of multiple clones or com-plex rearrangements. They are observed in 10–20% of pa-tients with primary MDS and up to 90% of patients with t-MDS (Olney and Le Beau, 2001). Most patients with com-plex karyotypes have abnormalities of chromosomes 5 and/or 7, together with other abnormalities. Complex karyo-typic abnormalities are associated with poor prognoses.

Abnormalities of chromosome 7 . Monosomy or deletion of the long arm of chromosome 7 is present in about 5% of de novo MDS and 55% of t-MDS (Heim and Mitelman, 1995; Sole et al., 2000). It is characterized by disease pro-gression and poor prognosis.

Intermediate prognosis group The remaining 14–17% of patients with various single

and double abnormalities not included in the other two groups were included in this group.

Trisomy 8 . About 10% of patients with MDS have trisomy of chromosome 8 (Morel et al., 1993; Vallespí et al., 1998; Sole et al., 2000). It is also associated with other recurring abnormalities of known prognostic significance.

Other abnormalities Rearrangements of 11q23 . About 5% of MDS patients re-

veal abnormalities involving band 11q23. It frequently oc-curs as part of a complex karyotype and is associated with other abnormalities, usually monosomy 7 or deletion of 7q. Prognosis is poor with progression to AML in 20–30% of cases (Bain et al., 1998).

Deletion of 17p . Rearrangements resulting in loss of the short arm of chromosome 17 have been seen in about 5% of MDS (Lai et al., 1995b; Soenen et al., 1998). Loss of 17p oc-curred through monosomy, deletion, unbalanced translo-

cations between 17p and another chromosome or isochro-mosome formation. Most of the patients had additional complex cytogenetic findings. Abnormalities involving chromosomes 5 and 7 were frequently present. Clinically, patients with 17p deletion had aggressive disease with poor response to therapy and short survival.

Conclusion

Cytogenetic and FISH analyses have elucidated the basic genetic and clinical heterogeneity of the various leukemias and lymphomas. Recurrent cytogenetic abnormalities have been shown to be associated with specific clinical charac-teristics and treatment outcomes in most hematological malignancies and their identification has had critical prog-nostic value by directing therapeutic decisions. FISH pan-els, with their improved detection rate of the recurrent aber-rations, have become established both as a means of identi-fying specific chromosomal abnormalities, and as genetic indicators of prognostic outcome and are regularly utilized in the initial assessment of a patient, particularly when the cytogenetic results are normal. They are also used to differ-entiate the heterogeneous nature of the leukemias, mani-fested by the different genetic subtypes. Evaluation of these and the monitoring of evolving cytogenetic abnormalities throughout the course of the disease allows clinicians to treat patients with more effective and targeted therapeutic regimens and assess their responses more effectively.

The significance of many abnormalities, particularly the less frequent ones, is currently unknown and most have been combined into single prognostic categories. Charac-terization of these and new genetic abnormalities that are still being discovered will further contribute to the develop-ment of therapeutic agents designed to target the unique genes involved and evaluation of their clinical outcome will help define their prognostic significance.

Routine cytogenetic analysis, however, still remains an irreplaceable procedure in the initial workup of a patient, because it provides a comprehensive representation of the spectrum of abnormalities. The strategy of using both pro-cedures together is highly informative and sensitive, and recommended.

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FISH panels for hematologic malignancies