Leukemia and Lymphoma. Vol. 17. pp. 361-366 Reprints available directly from the publisher Photocopying permitted by license only

0 1995 Harwood Academic Publishers GmbH Printed in Singapore

Molecular Analysis of the 5q- Chromosome LALITHA NAGARAJAN

Department of Hematology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA

(Received July 15. 1994)

Acquired interstitial deletions of the long arm of chromosome 5, are seen in anomalies of the myeloid cells. The refractory anemia (RA) or 5q- syndrome, in which the erythroid and megakaryocytic lin- eages are predominantly affected, is a relatively indolent clinical entity distinguishable, from the con- stellation of preneoplastic myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) with trilineage involvement. Recent molecular evidence suggests that the critical region of 5q deletion in MDSIAML resides in the D5S89 locus, which is proximal (centromeric) to the mini- mal region of loss in the 5q- syndrome RA. The invariable loss of the D5S89 locus in MDS/AML qualifies it for the MDSlAML tumor suppressor locus. The telomeric 5q3 I gene governs erythroid and megakaryocytic differentiation and can be termed the RA locus. Isolation and characterization of these genes will lead to an understanding of molecular mechanisms underlying normal hematopoiesis and leukemic transformation.

KEY WORDS: Chromosome 5q3 I refractory anemia myelodysplasia tumor suppressor loci

INTRODUCTION

The 5q- chromosome was first identified in patients with refractory anemia, in 1974, a time when the Philadelphia chromosome was the only known karyotypic anomaly as- sociated with myeloid disorders. I The anomaly was re- stricted to the myeloid lineage of these patients suggesting that the interstitial 5q deletion occurs in a myeloid prog- enitor cell. Subsequent studies demonstrated that the 5q- chromosome was also associated with myelodysplasia (MDS) and acute myelogenous leukemia (AML) and the limits of the cytogenetic deletion varied from patient to patient.2-7 However, a region of consistent loss between bands 5q23-3 1, was observed in all the patients.5 These observations led to the suggestion that loss of a critical gene from 5q, uncovers an inactivating mutation on the remaining allele. This phenomenon is explained by the two-hit model which predicted the existence of tumor sup- pressor genes.8

5q- Chromosome is associated with the indolent refractory anemia, preleukemic MDS and AML

The transfusion-dependant RA or 5q- syndrome, typically seen in elderly women is a clinical entity, readily distin-

Address for correspondence: Lalitha Nagarajan 36 I

guishable, from the constellation of other myeloid disor- ders designated myelodysplastic syndrome [MDS: re- fractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia in transformation (RAEBT)], and acute myeloge- nous leukemia (AML). Therapy induced MDS (t-MDS) and AML (t-AML) are common in patients treated for other forms of cancer. Fifty percent ofpatients with t-MDS or t-AML present with a 5q- chromosome which is a marker for poor prognosis in MDS and AML; AML pa- tients with no gross cytogenetic anomalies (diploid) have a better prognosis than MDS patients with 5q- chromo- some, despite the higher leukemic blast counts of the for- mer.'.lo

Two distinct phenotypes are associated with 5q- chromosome. Is the smallest region of overlap (SRO) common to both the 5q- syndrome and MDS/AML or is there more than a single critical gene on 5q31?

Patients with 5q- syndrome refractory anemia (RA), have a mild increase in erythroblasts (< 5%) and megakaryocytes; the 5q- chromosome is the sole cytoge- netic anomaly. In contrast, patients with MDS and AML harbor additional karyotypic abnormalities and, the bone marrow reveals anomalies of all the lineages with ery- throid hyperplasia, megaloblastoid features and increase

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362 L. NAGARAJAN

in immature granulocytes.11 The striking distinction be- tween the 5q- syndrome RA and other forms of MDS/AML is that the former has a relatively indolent and stable clinical course while the latter has a poor progno- sis. Furthermore, 5q- syndrome RA is uncommon among patients with t-MDS.

There are no apparent cytogenetic differences between the 5q- chromosomes of RA, MDS and AML. A retro- spective cytogenetic study of 50 patients with interstitial deletions of chromosome 5 identified at least four types of cytogenetically identifiable deletions: (i) 5q dell3-33, (ii) 5q de131-35, (iii) 5 delq22-33 and (iv) 5q del 13-35. Using high resolution banding techniques, 5q3 1 has been identified as the most common cytogenetically identifi- able segment, which is deleted in 91 out of 93 patients with 5q deletion.12 This region of overlap spans 17Mbs of DNA and is thus much larger than typical deletions seen in inactivation of well characterized tumor suppressor genes (retinoblastoma and familial and sporadic forms of the Wilm's tumor and neurofibromatosis).

Whether the smallest region of loss is common to all the phenotypes remains to be established. As a corollary, whether a single critical gene is inactivated in these dis- eases is unknown. The molecular cloning of a gene fusion, between the protein kinase domain of the platelet derived growth factor receptor (PDGFR p), and a novel helix loop helix transcription factor gene rel, is an illustration of a correlation between chronic myelomonocytic leukemia (CMMoL), a specific subset of MDS and a unique gene rearrangement. 13

Molecular studies of the 5q anomaly began in 1985, when the gene for the cytokine GMCSF, was localized to the region of loss in the 5q- chromosome.14 The cellular homolog of the feline oncogenic virus, v-fms, encoding the receptor for the cytokine CSFl (CSFIR), was mapped to the portionof 5qdeleted in patients.15 Molecular cloning and chromosomal mapping of the genes for 1153, IU, IL5, and ZL9 revealed evolutionary clustering of the interleukin genes in the most frequently deleted region; additionally the genomic organization of these genes is very similar suggesting that they evolved from a common ancestor. Thus, the isolation of interleukin genes pointed to a chro- mosomal lochs of tremendous importance in normal hematopoietic growth and differentiation and provided serendipitous molecular 1andmarks.If-19 Localization of other genes which are tightly regulated during myeloid differentiation, namely the monocyte antigen CD 14. the zinc finger transcription factor EGRI, which is an early growth/differentiation response gene and the Interferon Response Factor ( I R F I ) , within 5q, contributed additional reagents.2w22

Within the past year, considerable progress has been

made in the definition of the minimal region of overlap, employing Fluorescent insitu hybridization (FISH), Yeast Artificial Chromosome (YAC) cloning and irradiation re- duced hybrid mapping techniques. Figure 1 depicts the consensus order of genes mapped to 5q3 I , a region most frequently deleted in patients with 5q- chromosome.

Molecular evidence for the presence of more than a single critical locus

AML: Restriction fragment length polymorphism analy- ses demonstrated invariable loss of an informative poly- morphic locus DSS89 from the 5q- chromosome of paiients with AMUMDS.23-25 Radiation hybrid mapping localized the DSS89 locus between the IL9 and EGKl genes.25 FISH of 16' cases of MDS/AML with overlap- ping 5q3 1 deletions, using a panel of 5q3 1 probes, iden-

MDS/AML 5q31 1 2 3 4

16.1 n I S 3

11.1 11.1 11.1 11 11

11.1 11.1

12 11.1 11.1 11.3

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11

12 21.1 13 2

51.1 11.2 11.3

31 3 l . I

3l.3

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13.2

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; 51

i.

IRF1

GMCSF

I L9

DSS89

EGRl

D5S166

CD14

FG FA

GRL

CSFlR

SPARC

GLUHl

1

. . - 1 I

5q- RA 5 6

T ' T Figure 1 Consensus Map of the critical loci on human chromosome 5q31 and limits of the interstitial deletions seen in patients. a. Order of genes are as reportedelsewhere. Genes and loci are denoted on the right. lL5, interleukin 5; ; IRF-I. interferon response factor I;GMCSF. gran- ulocyte macrophage growth factor; IL9, interleukin 9 EGR I , early growth response gene 1; CD14. Monocyte antigen; FGF A. Acidic Fibroblast Growth Factor; GRL, Glucoconicoid receptor CSF I R, colony stimulating factor1 receptor. SPARC- Osteonectin; GLUH I glu- tamate receptor; and NKSFI-fLI2 subunit. The distance hetween IL9D5S89EGRI and CDI4/FGFA/GRL are denoted in centiray (cR) units ( I cR z 34kbp). Patients characterized to retain proximal and dis- tal loci within the minimal region overlap have been previously de- scribed. Patients 1 and 2 were described by Le Beau er a/.,?6 patients 3 and 4 by Nagarajan el ~ 1 . 2 5 3 2 and patients 5 and 6 by Boultwood er u/." Lines denote loci retained.

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MOLECULAR ANALYSIS OF THE Sq-CHROMOSOME 363

tified the loci telomeric to lL9 and centromeric to the anonymous locus D5S166 reside within the smallest re- gion of overlap. A case of de novo AML retained lL9, and all of the centromeric probes used; a case of t-AML re- tained DSS166 and all of three other telomeric loci. E G R l , was the only locus consistently deleted from the 5q- chro- mosome of all of these patients; these findings imply that D5S89, resides within the minimal region of loss.26

5q- Syndrome: A survey of patients with 5q- syndrome RA. by Boultwood et al, identified two patients with un- characteristically small interstitial deletions (del. 5q3 1-33).27 One of these patients, had previously been re- ported to have a homozygous deletion of the C S F l R gene.?* The region of loss in the second patient, which was recognized to be the smaller of the two, resides between the gene for glucocorticoid receptor (GRL) and the gene encoding the ysubunit for interleukin IL 12 (NKSFI), with apparent retention of all the centromeric and telomeric

The physical distance between lL9 and CSFl R genes is estimated to be 7 Mb. by radiation hybrid map- ping,29*30 whereas inter phase FISH experiments indicate this distance to be < 5 Mb.31

Interstitial loss of 5q sequences is not contiguous in t- MDS. Sequences from the D5S89 and C S F l R loci are deleted from the 5q- chromosome of a patient (t-RAEBT) with retention of the intervening region: A case of t- RAEBT with del5q( 1 1-3 I), and impaired neutrophil mat- uration was studied in detail by us.32This patient, who w p treated with alkylating agents over a period of five years for mesothelioma of the lung, presented with 2 1 % blasts in the bone marrow. There were numerous cytogenetic abnormalities, suggestive of karyotypic instability. A 5q- chromosome was detected in 90% of the metaphases an- alyzed.

FISH analysis with 5q3 1 specific probes, demonstrated that the interstitial aletion on the 5q- chromosome is not contiguous in this patient. Three FISH probes were em- ployed in this study: (i) sequences representing a 300 kbp YAC spanning DSS89, (ii) sequences representing a radi- ation hybrid spanning D5S89 and EGRl but neither of the flanking markers ( lL9 and CD14) . and (iii) two cosmids spanning the CSFlR locus. The 5q- chromosome has lost the D5S89 and CSFlR loci while retaining some of the, interstitial sequences, derived from a radiation hybrid spanning D5S89 and E G R l . These results suggest that there are two distinct critical loci on 5q3 1 and that the in- terstitial deletions are not contiguous in cases of t- MDS/AML. Future studies on t-MDS/AML cases treated with alkylating agents for primary malignancies will allow us to determine whether the multiple, noncontiguous in- terstitial deletions seen in this case are frequent in t- MDS/AML.

Development of a sensitive polymerase chain reaction (PCR) to detect allele loss in 5q- chromosome

In contrast to the preneoplastic lesions in adenomatous polyposis coli or neuro fibromatosis, the dysplastic clone associated with 5q deletion in RA and MDS lacks growth potential. Consequently the material available for molec- ular analyses is limited. Other technical impediments in- clude a paucity of highly polymorphic loci on the distal long arm of chromosome 5 and technical limitations in separating the affected cells from the normal population. Thus, the time-honored loss of heterozygosity analyses for polymorphic loci, which is used to define the smallest re- gion of deletion in molecular terms, could not be readily applied to the 5q- chromosome.

Tandemly repeated dinucleotide (dC-dA)n and (dG- dT)n sequences exhibit high variability in the number of copies of the repeats in the normal human population. The alleles can be amplified by PCR, employing unique primers flanking the repeats and resolved on strand-sepa- rating denaturing polyacrylamide gels.32 Taking advan- tage of a highly polymorphic dinucleotide repeat within the interleukin 9 (IL9) gene on 5q3 1, we have developed a PCR based assay to detect loss of chromosome 5q31 l0ci.33 Allele loss is detected by comparing the intensities of the amplification from ficoll buoyant and pelletted pe- ripheral blood fractions of patients with RA or MDS. In cases of RAEBT and AML, the Ficoll-buoyant mononu- clear blast population is compared with short-term cul- tured T cells. These studies revealed a loss of the IL9 gene from the 5q- chromosome, in 10 out of 12 cases of MDS/AML patients.

The peripheral blood neutrophils are derived entirely from the 5q- clone in 5q- syndrome RA. Dinucleotide poly- morphism analysis has allowed us to study the lineages involved in the 5q- syndrome.33 A transfusion-dependent RA patient who had a 5q- chromosome as the sole anom- aly with normal peripheral blood neutrophil and mono- cyte counts and decreased erythroid cells and platelets revealed that the peripheral blood neutrophils are derived entirely from the 5q- chromosome. Thus the neutrophil maturation pathway seems unaffected by the deletion of part Of 5q, in these cases. Other studies have identified ex- pression of the monocyte antigen CD 14 and the leukocyte antigen CD45 on the surface of the monocytes of RA cases suggesting that the myelomonocytic differentiation in these patients is not blatantly influenced.34 Thus, the in- dolent 5q- syndrome represents impairments only in the megakaryocytic and erythroid maturation, whereas MDS and AML with 5q deletions exemplify an arrest in a tri- lineage precursor. These findings underscore the major

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364 L. NAGARAJAN

differences between the 5q- syndrome RA and other myeloid anomalies with the 5q- chromosome.

5q- chromosome is seen during the evolution of AML

The D5S89 locus, which resides within the critical region of overlap seen in MDS/AML cases, was originally iso- lated and characterized because of it's molecular re- arrangement on a marker chromosome of the AML cell line HL60. The patient from whom the HL60 cell line, was derived was monosomic for human chromosome 5 at di- agnosis and had a marker chromosome containing 5q ma- terial.35 Southern blotting analysis of HL60 cells revealed both a normal GMCSF locus and a rearranged GMCSF locus. Molecular cloning of the rearranged GMCSF locus led to the characterization of an intra chromosomal Long Interspersed Nuclear Element (LINE) mediated insertion of the GMCSF locus remnant, into a novel polymorphic locus, which was designated DSS89.23 These rearrange- ments are identical in both early passage (19") and late passage (>200") HL60 cells in culture, suggesting that these genetic alterations are stable and may have occurred in the patient's leukemic cells.

What is the likely scenario leading to these complex deletion coupled rearrangements? In developing a model the following series of events will have to be taken into account: (i) LINE insertion into 3' portion of the GMCSF gene, (ii) Truncation of GMCSF coding sequences and tag- ging of the 3' GMCSF flanking sequences by the L1 se- quence, (iii) Insertion of the LINE + GMCSF locus tag into the D5S89 locus with loss of loci between GMCSF and D5S89.

The elegant murine hematopoietic stem cell model de- veloped by Stocking er nl.,36 to unravel molecular events leading to factor independent growth provides valuable clues. Clones of murine hematopoietic stem cells in cul- ture which escaped the requirement for exogenous inter- leukins, evolve due to transcriptional activation of the GMCSF gene by intracisternal A particle insertion in the 3' untranslated region of the gene.36 In an analogous sce- nario the LINE insertion 3' of the GMCSF gene could have provided an autocrine GMCSF stimulus to a clone in the preneoplastic state or early stages of transformation. Accumulation of additional molecular events (e.g., MYC amplification), could circumvent the need for autocrine GMCSF. During the evolution of the disease clones with subsequent loss/inactivation of genes governing myeloid growth and differentiation would have a proliferative ad- vantage. Finally, the loss of sequences between GMCSF and D5S89 would lead to deletion of the MDS/AML tumor suppressor gene which was accompanied by insertion of the truncated GMCSF by the LINE sequence into the telomeric D5S89 locus.

An AML patient, who acquired a 5q- chromosome dur- ing relapse lends further support to this model. This pa- tient presented with the cytogenetic anomaly t(8;2 l), which may have been the underlying transforming anom- aly. The relapse samples had a 5q- chromosome in addi- tion to t(8;21). Our studies had established loss of the D5S89 locus in this patient, with retention of two alleles of IRF-1.25 These results suggest that a critical 5q31 gene(s) regulates expression of several genes associated with normal myeloid maturation and that loss of this gene(s) would lead to dedifferentiation of the transformed clone.

The MDWAML tumor suppressor gene and the RA gene are on 5q31

Developments in the molecular analyses of the 5q- chro- mosome suggest that the proximal gene encoded in the DSS89 locus governs myeloid growth and differentiation; loss of function of this locus plays a role in RAEBT and AML with poor prognosis. Thus the phenotype associated with loss of D5S89 implicates this region as the MDS/AML tumor suppressor locus. The distal region in the vicinity of the CSFIR gene is a regulator of erythroid and megakaryocytic differentiation and can be termed the RA locus (Fig. 2).

Isolation of the critical 5q genes encoded within the two 5q loci will serve as the first step in understanding the in- terplay between these and several other regulatory genes on 5q (IRFI, EGRl and CD14) and their effects on the phenotypic heterogeneity seen in patients with deletions on the long arm of chromosome 5.

Oiatal 5q31 gono

platelets

eorinophila

basophils Proximal 5q91 gem

Figure 2 Model for the role of two 5q3 I regulatory genes which gov- em hematopoiesis. The 5q- deletion occurs in the erythroid/myeloid progenitor. Loss of the distal 5q31 gene, in the CSFlR locus, impairs erythroid and megakaryocytic maturation, leading to refractory anemia (5q- syndrome). Loss of proximal gene in the D5S89 locus affects gran- ulocytic growth and differentiation, resulting in pre neoplastic MDSIAML.

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MOLECULAR ANALYSIS OF THE 5q-CHROMOSOME 365

Recent findings on myogenic differentiation in mice in which a single or both alleles of two regulatory gene(s) (my0 D and myf-5), are inactivated illustrate the com- plexity of interactions that occur, between genes regulat- ing the differentiation cascade.37 Mice which lack both the alleles of my0 D have a compensatory increase (three-fold) in myf-5expression and have no gross deficiencies in myo- genic differentiation. This compensation requires two al- leles of myf-5; however if these mice have lost one allele of the myf 5 gene, they are severely impaired. The phe- notypes associated with 5q- syndrome RA and MDS/AML are discernable; but the mechanisms underlying the het- erogeneity among the MDWAML patients with 5q dele- tions is poorly understood. Interactions between the MDWAML and RA tumor suppressor genes similar to those of myoD and myf-5 may not be ruled out. Molecular cloning of the MDS/AML tumor suppressor gene and the RA gene will be a crucial step in unraveling the intrica- cies of myeloid differentiation.

Acknowledgements Work in the author’s laboratory was sup- ported by grants f rom NIH (Ca 55164) and American Cancer Society (DHP44). I thank Drs. Grady Saunders and Nat Sternberg for critical reading of the manuscript and Jiri Zavadil for assistance in the art work.

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