Clonal heterogeneity in the 5q- syndrome: p53 expressing progenitors prevail during lenalidomide treatment and expand at disease progression

Clonal heterogeneity has not been described in patients withmyelodysplastic syndrome with isolated del(5q), for which lenalidomidehas emerged as a highly potent treatment. However, transformationto acute myeloid leukemia is occasionally observed, particularlyin patients without a cytogenetic response to lenalidomide.We performed molecular studies in a patient with classical 5q-syndrome with complete erythroid and partial cytogenetic responseto lenalidomide, who evolved to high-risk myelodysplastic syndromewith complex karyotype. Immunohistochemistry of pre-treatmentmarrow biopsies revealed a small fraction of progenitors withoverexpression of p53 and sequencing confirmed a TP53 mutation.TP53 mutated subclones have not previously been described inmyelodysplastic syndrome with isolated del(5q) and indicatesa previously unknown heterogeneity of this disease. The aberrantsubclone remained stable during the treatment with lenalidomideand expanded at transformation, suggesting that this pre-existingcell population had molecular features which made it insensitiveto lenalidomide and prone to disease progression.

Key words: resistance, del(17p), thalidomide analog.

Introduction

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Introduction

The immunomodulatory drug lenalidomide is a potent treatmentfor low-risk myelodysplastic syndrome (MDS) with a karyotypeincluding the deletion of 5q31 (del[5q]), with 67% achievingtransfusion independency, and 45% obtaining complete cytogeneticremission.¹ However, 50% of responders relapse within two years,and the estimated risk of leukemic transformation at ten yearsis 15% for cytogenetic responders and 67% for patients withouta cytogenetic response.²

Design and Methods

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Design and Methods

Patients and treatment
A patient with classical 5q- syndrome (MDS with isolated del[5q]according to the WHO 2008 classification)³ received lenalidomidewithin a clinical trial (Celgene MDS-004). The clinical andlaboratory studies were approved by the Ethical Committee forResearch at the Karolinska Institutet, Stockholm, Sweden. Informedconsent was obtained from the patient.

Bone marrow assessment and immunohistochemistry
Histopathology and morphology were assessed on consecutive bonemarrow (BM) samples. Immunohistochemistry on paraffin-embeddedsections was performed using mouse monoclonal antibodies: p53(clone DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA);CD34 (DakoCytomation, Glostrup, Denmark). Ten normal BMs werestained and demonstrated p53 expression in less than 0.01% ofcells.

Chromosome banding analysis
After 20-48 h of culture, metaphases of BM were prepared andfluorescence R-banding was performed.⁴ Karyotypes were describedaccording to the International System for Human CytogeneticNomenclature.⁵

Fluorescence in situ hybridization (FISH) and M-FISH
Interphase FISH was performed using a probe for the locus 5q31.⁶ Depending on the cytogenetic aberrations detected, probes(Abbott, Wiesbaden, Germany) were applied for the MLL-locus(11q23), RB1-locus (13q14), TP53-locus (17p13), BCL2-locus (18q21),and AML1-locus (21q22). At least 200 nuclei were evaluated.M-FISH analysis was carried out as previously described.⁶

Cells and cultures
BM sampling for in vitro culture was performed pre-lenalidomideand at time of disease progression. CD34⁺ progenitors were selectedfrom BM mononuclear cells and cultured for seven days ±lenalidomide 10 µM, as recently described.⁷

Gene expression profiling and gene sequencing
RNA and DNA were extracted from cultured progenitors at day7. Gene expression profiling analysis was performed as describedelsewhere.^7,8 Pathway analysis was performed using IngenuityPathway Analysis software (Ingenuity Systems, Redwood City,CA, USA). DNA-sequences spanning exons 5–8 of TP53 wereamplified by polymerase chain reaction (PCR), using publishedprimer sequences.⁹ PCR products were sequenced using the BigDyeTerminator v1.1 kit (Applied Biosystems).

Results and Discussion

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Results and Discussion

Clinical observations
A 76-year old woman was diagnosed with 5q- syndrome in April2004. She was transfusion-dependent up-front (2 units of redblood cells per month), and had a transient response to EPO.Lenalidomide treatment was started in February 2006, leadingto a complete ery-throid response, and a reduction in the del(5q)clone from 75% to 25% by FISH. G-CSF was given during the firstmonths. The transfusion-need recurred following pneumonia inOctober 2007; however, neutrophil and platelet counts were stillnormal. In November 2007, 22 months after start of treatment,BM examination showed 9% blasts and cytogenetic analysis demonstrateda large clone with complex karyotype, including del(5q). ByFISH, a loss of the TP53 locus at 17p13 and a gain of the MLL-and the RB1-loci could be confirmed (Table 1, Figure 1). InDecember 2007 she was started on 5-azacytidine and became transfusionindependent, although there was no decrease in bone marrow blasts.After 8 cycles she progressed to full leukemia and succumbedto an infection.

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Table 1. Summary of clinical data and laboratory investigations.

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Figure 1. (A) Karyogram of a metaphase at time of progression shown by M-FISH. (B) Partial karyograms of the structural aberrant chromosomes 11, 16 and 17 shown in detail. After M-FISH the additional chromosomal material in the short arm of the derivative chromosome 11 could be identified as material of chromosome 16: der(11)t(11;16)(p11;?). The additional chromosomal material in the long arm of the derivative chromosome 16 and the additional material in the short arm of the derivative chromosome 17 were identified as material of chromosome 11: der(16)t(11;16)(q12;q12) and der(11;17)(p10;q10). Immunohistochemistry demonstrated (C) a population aberrantly over-expressing p53 already pre-treatment, and (D) a clear increase of this population at disease progression.

Cell culture and gene expression
Pathway analysis based on the gene expression profiles of culturedcells (86–96% del[5q] by FISH) demonstrated altered apoptosisand integrin signaling at treatment failure compared pre-treatment,which may reflect a more aggressive disease and an altered interactionbetween MDS progenitors and stroma.

We also assessed the effect of lenalidomide on mRNA expressionof SPARC and Activin-A, based on data from a previous study.⁷ Both genes were up-regulated in post-treatment samples (3.6–7.1and 2.5–5.1 fold, respectively), similarly to our findingsin lenalidomide naïve samples.⁷

As the complex karyotype at progression included trisomy 13we assessed the expression level for the FLT3 gene, locatedat 13q12. There was an upregulation of FLT3 at the time of progression,however, from a low pre-treatment level.

Mutated TP53 pre-treatment
Before treatment with lenalidomide, 1–2% of the BM progenitorsover-expressed p53, which may indicate a mutation in the TP53tumor suppressor gene.¹¹ TP53 plays a crucial role in genomicintegrity and stability.¹² Interestingly, this subclone expandedin parallel with the blast count at disease progression (Table 1, Figure 1). TP53 was sequenced in pre- and post-treatmentsamples. Before treatment, an A>G mutation in exon 5 (Y163C)was identified at a similar levels as the wild-type gene. Attime of progression the mutated gene dominated, consistent withthe cytogenetics demonstrating a complex karyotype includingdel(17p13) resulting in loss of the one TP53 allele (Table 1).

TP53 mutations are exceedingly rare in the 5q- syndrome,¹³ however,they frequently occur in MDS with complex karyotypes includingdel(5q) or in therapy-related MDS, invariably implying a pooroutcome,^14,15 The frequency of small fractions of BM progenitorswith abnormal p53 expression, which may fall below the detectionthreshold for a mutational analysis, is unknown in MDS withisolated del(5q).

Potential prognostic implications of p53 expressing subclones pre-treatment
The TP53 mutated clone in our case was present before treatmentwith lenalidomide, remained stable despite a partial cytogeneticresponse, and expanded at progression. We argue that this cloneconsisted of cells with inherent genomic instability which increasedtheir probability of acquiring molecular features making themless sensitive to lenalidomide. The p53 expressing clone expandedin parallel to the blast count at time of transformation, andthe complex karyotype evolving included deletion of the TP53locus at 17p13 in addition to trisomy 13, incorporating theFLT3 gene, and rearrangements leading to a gain of the MLL locuson chromosome 11. Consequently, inactivation of p53 and acquisitionof additional genetic aberrations are likely to have contributedto progressive disease in this patient. Moreover, the presenceof trisomy 13 at time of disease progression during lenalidomidetreatment is in contrast to a recent report suggesting thatAML patients with trisomy 13 may respond particularly well tolenalidomide.¹⁶

Leukemic evolution occurs in around 10% of patients with classical5q- syndrome (blasts <5% and an isolated del[5q]) duringthe natural course of the disease, while the risk of progressionis higher in the presence of additional cytogenetic aberrationsor a blast increase.¹⁷ The cytogenetic characteristics at timeof transformation have not been well studied. A preliminaryreport indicated that 7 of 22 (32%) MDS patients with less than10% BM blasts and a karyotype including del(5q) receiving lenalidomidedeveloped complex karyotypes at time of progression, at a medianof 44 months from diagnosis.¹⁸ Of the 7 patients who progressed,2 patients had a true low-risk 5q- syndrome, while 5 had a blastcount 5-9% or del(5q) with one additional chromosomal abnormality.In addition, a recent report described a patient with classicallow-risk 5q- syndrome that transformed to AML with a highlycomplex karyotype after eight months of treatment with lenalidomide.¹⁹ The European Medicines Agency (EMEA) recently advised againstthe approval of lenalidomide in Europe for patients with low-riskMDS and del(5q) stating that a treatment-associated increasein the risk of leukemic transformation could not be excluded.²⁰

It is conceivable that lenalidomide may suppress a favorableclone and allow expansion of a more malignant subclone; however,it is equally conceivable that the potent effect of lenalidomidemay delay progression. This question is best addressed in arandomized trial. A first step could be to analyze the associationof p53 overexpression and TP53 mutation with the probabilityof disease progression in an expanded cohort of MDS del(5q)patients. This will not answer the question whether lenalidomideaffects the natural course of the disease; however, it may identifya subgroup of patients at higher risk of transformation whoshould be carefully monitored and in which other treatment modalitiesshould be explored. The presence of easily detectable subcloneswith inactivated p53, and thereby a more malignant potential,represents a novel concept in low-risk MDS and may have importantprognostic implications.

Acknowledgments

we express our warmest gratitude for excellent technical supportto Lalla Forsblom (cell separation and culture) and Monika Jansson(FISH analysis) at the Karolinska Institutet, Department ofMedicine, Division of Hematology, to M. Hagedorn, M. Schoop,J. Espenkötter, R. Rodde, W. Arndt, and M. Meyer (cytogenetics)at Hannover Medical School, and Cristina Fernández-Santamaría(TP53 sequencing), The LRF Molecular Haematology Unit, NDCLS,John Radcliffe Hospital, Oxford. This work was supported bythe Competence Network "Acute and chronic leukemias". Finally,we thank Dr. Robert Knight, Celgene, for allowing us to usecytogenetic data obtained within the Celgene MDS-004 clinicaltrial.

Footnotes

Funding: the work was financially supported by grants from TheInternational MDS Foundation (MJ), Swedish Cancer Foundation(070678;EH-L), Swedish Medical Research Council (K2005-71X-15273-01A;EH-L), the Cancer Society in Stockholm (051143; EH-L), and theLeukaemia Research Fund of the U.K. (JB).

Authorship and Disclosures

Designed research: MJ, LS, GG, APo, BS, EHL; managed the patient:MJ and EH-L; generated and analyzed data: MJ, LS, AP, GG, JSW,JB, APo, BS, EH-L; wrote the paper: MJ, LS, GG, APo, BS, EH-L.

MJ received lecture fees from Celgene and is co-investigatorof Celgene MDS-004 trial. BS and GG are part of the centralreference laboratory for cytogenetics in the Celgene MDS-004trial. EHL is the Swedish principal investigator in the CelgeneMDS-004 trial.

Received for publication May 17, 2009. Revision received June 12, 2009. Accepted for publication June 16, 2009.

References

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