Detection of cryptic chromosomal lesions including acquired segmental uniparental disomy in advanced and low-risk myelodysplastic syndromes

Experimental Hematology 35 (2007) 1728–1738

Detection of cryptic chromosomal lesions including acquired segmental uniparental disomy in advanced and low-risk myelodysplastic syndromes Lukasz P. Gondeka, Abdo S. Haddada, Christine L. O’Keefea, Ramon Tiua, Marcin W. Wlodarskia, Mikkael A. Sekeresb, Karl S. Theilc, and Jaroslaw P. Maciejewskia,b a Experimental Hematology and Hematopoiesis Section; bDepartment of Hematologic Oncology and Blood Disorders, Taussig Cancer Center; cDepartment of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio., USA

(Received 1 May 2007; revised 30 July 2007; accepted 7 August 2007)

Objectives. Using metaphase cytogenetics (MC), chromosomal defects can be detected in 40% to 60% of patients with myelodysplastic syndromes (MDS); cytogenetic results have a major impact on prognosis. We hypothesize that more precise methods of chromosomal analysis will detect new/additional cryptic lesions in a higher proportion of MDS patients. Methods. We have applied single nucleotide polymorphism microarrays (SNP-A) to perform high-resolution karyotyping in MDS to determine gene copy number and detect loss of heterozygosity (LOH). Results. Using this method, chromosomal defects were found in 82% of MDS patients vs 50% as measured by MC; lesions were present in 68% of patients with normal MC, while in 81% of those with abnormal MC, new aberrations were found. In addition to gains or losses of chromosomal material, areas of LOH due to segmental uniparental disomy were found in 33% of patients. Conclusion. SNP-A findings demonstrate that chromosomal lesions are present in a much higher proportion of patients than predicted by traditional cytogenetics. These lesions may reflect an underlying generalized chromosomal instability in MDS. Additional previously cryptic defects may explain the clinical variability of MDS. New lesions may have important prognostic implications, suggesting that, in the future, SNP-A–based karyotyping may complement MC in laboratory evaluation of MDS. Ó 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Myelodysplastic syndromes (MDS) are associated with peripheral cytopenias due to ineffective and dysplastic hematopoiesis. The clonal nature of MDS has been confirmed by X-chromosome inactivation and cytogenetic findings of nonrandom, acquired chromosome abnormalities [1,2]. In MDS, certain chromosomal lesions are associated with distinct clinical phenotypes and have a significant prognostic impact [3,4]. While defects are found in only 40% to 60% of MDS patients when metaphase cytogenetics (MC) is applied [5], chromosomal changes may be present in a higher proportion, if not in all patients, but our ability

Offprint requests to: Jaroslaw P. Maciejewski, M.D., Ph.D., Experimental Hematology and Hematopoiesis Section, Taussig Cancer Center R-40, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195; E-mail: [email protected]

to detect them is limited by the low resolution of current metaphase banding techniques. Karyotypic abnormalities may determine phenotypic properties of the affected clones. For example, duplication of chromosomal material may lead to overexpression of oncogenes. In contrast, deletions may result in decreased expression of tumor suppressor proteins or loss of heterozygosity (LOH) in genes for which only a defective allele is retained. LOH has been described in many malignancies [6,7], and can occur through several different mechanisms. In addition to loss of chromosomal material, LOH may be due to uniparental disomy (UPD) resulting from mitotic recombination [8,9]. Such changes occur without a decrease in gene copy number, and are not evident by MC. UPD9p has been described in the context of polycythemia vera [10] as a mechanism resulting in homozygosity for the Jak2 mutation [11].

0301-472X/07 $–see front matter. Copyright Ó 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2007.08.009

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

Any improvements in the resolution of cytogenetic techniques are likely to have important clinical implications. New nonrandom lesions with clinical significance may be identified, and smaller cryptic lesions may turn out to have prognostic significance analogous to known larger defects of the corresponding chromosome. Finally, accessory, previously cryptic, defects may modify the phenotype produced by common, well-established aberrations. High-density arrays that contain probes specific for single nucleotide polymorphisms (SNPs) allow for whole genome scanning in genetic linkage and association studies [12]. However, SNP arrays (SNP-A) can also be applied for detection of copy number changes and copy-neutral LOH, and allow for genomic scan with increased resolution. The resulting DNA-based SNP ‘‘karyograms’’ facilitate detection of chromosome defects without the need for dividing cells, as in conventional cytogenetics. Genome-wide SNP maps can also be used to detect LOH occurring due to segmental, acquired UPD. Array-based karyotyping has been used for detection of genomic lesions in multiple myeloma [13] and acute myelogenous leukemia [14,15]. While applying SNP-A to study bone marrow failure syndromes, we found and reported, for the first time, that clonal UPD affecting various regions of the genome is particularly frequent in MDS [16]. MDS constitutes a very suitable target for application of SNP-A, because cytogenetic abnormalities are relatively frequent and have prognostic and clinical implications. Furthermore, most of the chromosomal aberrations in MDS are unbalanced and, therefore, amenable to SNP-A–based diagnostics. Identification of new lesions could refine prognosis

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in a significant proportion of MDS patients with normal cytogenetics, or in those with established chromosomal defects. We hypothesized that SNP-A karyotyping would confirm known cytogenetic abnormalities and reveal new previously cryptic genomic abnormalities.

Materials and methods Patients A total of 106 bone marrow aspirates and 7 blood samples were analyzed. There were 33 controls and 74 patients; 72 had MDS and 2 had other causes of anemia. Informed consent was obtained according to the protocols approved by the Cleveland Clinic Institutional Review Board. Patients were grouped according to the World Health Organization classification [17] and the International Prognostic Scoring System [3]. Mean age of the MDS cohort was 69 years (range, 26–86 years). Aspirates obtained from 33 healthy individuals (mean age: 42 years; range, 27–61 years) were used as controls. Specimens from two patients with nonmalignant hematologic diseases (hypersplenism, thalassemia trait) served as hematologic controls (Table 1). Cytogenetic analysis Cytogenetic analysis was performed on marrow aspirates according to standard methods. Chromosome preparations were G-banded using trypsin and Giemsa (GTG) and karyotypes were described according to International System for Human Cytogenetic Nomenclature (2005) [18]. SNP-A analysis DNA from total bone marrow, blood, and granulocytes was extracted using the Puregene DNA Purification Kit (Gentra, Minneapolis, MN, USA). To study the germline genotype, T lymphocytes

Table 1. Clinical characteristics of patients participating in the analysis Metaphase karyotyping WHO

n

WHO subtypes (no. of patients)

Risk categorya

Normal

Abnormal

No growth

MDS (n 5 59)

72

RA (n 5 9) RCMD (n 5 11) MDS with isolated 5q-(n 5 2) RARS (n 5 6) RCMD-RS (n 5 8) Undetermined (n 5 2)b RAEB-1 (n 5 7) RAEB-2 (n 5 10) sAML (n 5 4) CMML-1 (n 5 4) CMML-2 (n 5 3) RARS-t (n 5 6) Normal (n 5 33) Pancytopeniac (n 5 2)

Low (n 5 38)

4 4 0 2 3 2 3 4 0 2 2 5 NA 2

5 5 2 3 5 0 4 6 2 2 1 1 NA 0

0 2 0 1 0 0 0 0 2 0 0 0 NA 0

MDS/MPD (n 5 13)

Controls

35

High (n 5 21)

Low High Low NA

sAML 5 secondary acute myeloid leukemia; CMML 5 chronic myelomonocytic leukemia; MDS/MPD 5 myelodysplastic syndrome/myeloproliferative syndrome; RA 5 refractory anemia; RAEB 5 refractory anemia with excess blasts; RARS 5 refractory anemia with ring sideroblasts; RARS-þ/RARS associated with marked thrombocytosis; RCMD 5 refractory anemia with multilineage dysplasia; WHO 5 World Health Organization. a Low risk (n 5 48): MDS/MPD included RA, RCMD, 5q, RARS, RCMD-RS, CMML-1, RARS-t, MDS/MPD-U with #5% of blasts (not present). Advanced (n 5 21): RAEB1/2, sAML, CMML-2 and MDS/MPD-U with O5% of blasts (not present). b Clinically suspected to have MDS without sufficient morphologic evidence. c One thalassemia trait, one hepatic cirrhosis.

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L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

(CD3þ) were isolated using immunomagnetic beads (Miltenyi Biotec, Auburn, CA, USA). The Affymetrix Gene Chip Mapping 50K Assay Kit (Affymetrix, Santa Clara, CA, USA) was used for analysis. Following XbaI digestion (New England Biolabs, Ipswich, MA, USA), fragmented DNA was ligated to XbaI adaptors using T4 ligase (New England Biolabs) followed by polymerase chain reaction (PCR) amplification. The PCR product was hybridized to the gene chip array, and analyzed by Gene Chip Scanner 3000 (Affymetrix). For validation of microdeletions and duplications, samples were rerun on a 250K SNP-A (250K Nsp GeneChip Mapping Array, Affymetrix). Microsatellite and gene copy number analysis Validity of SNP-A as a method of detection of deletions and UPD has been confirmed in previous studies [16,19]. In this study regions of LOH were also confirmed by microsatellite (MS) analysis. MS PCR, the equivalent to short tandem repeats analysis, was used. MS with a high-level heterozygosity were selected to generate informative results. Primer sequences for all MS were obtained from the National Center for Biotechnology Information database (available at: http://www.ncbi.nlm.nih.gov). Forward primers were modified at the 50 end with 6-carboxy-fluoroscine. DNA extracted from marrow and CD3þ cells was amplified and amplicons analyzed using ABI Prism 310 Genetic Analyzer (ABI, Foster City, CA, USA). For informative MS, loss of one of the alleles (decrease in the specific peak intensity) indicated LOH for the corresponding locus. Copy number determination was performed by MS analysis using a real-time TaqMan chemistry protocol [20]. For copy number determination, a quantitative PCR assay was designed with a universal TaqMan probe to match CA repeats. This probe was designed as a 21-bp oligomer containing GT repeats with FAM and Black Hole Quencher modifications on 50 and 30 ends, respectively. All reactions were performed in triplicate using the D12S1699 amplicon as control. Such an analysis was performed to confirm UPD (n 5 5) and deletions (n 5 2). Biostatistical evaluation Signal intensity was analyzed and SNP calls determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number was investigated using a Hidden Markov Model and Copy Number Analyzer for Affymetrix GeneChip Mapping 100K arrays (CNAG) [21]. Segmental LOH was identified using CNAG software by a statistical assessment of the likelihood that consecutive SNP loci would exhibit heterozygosity, given the corresponding allelic frequency of particular SNP in the control population. Lesions identified by SNP-A were compared with the Cancer Genome Anatomy Project database (available at: http:// cgap.nci.nih.gov). Two-sided Fisher’s exact test was used to analyze the difference between distributions of dichotomized variables among the groups.

Results Principles of SNP analysis SNP-A utilize evenly distributed tag-SNPs for genetic association studies. For the purpose of this study, we used samples for which O90% SNPs could be genotyped (call rate).

By analysis of 1204 heterozygous SNP calls within the X chromosome in 42 males, the accuracy of the array was estimated to be 99.42%. Bone marrow aspirates from normal individuals were used to define pathogenic chromosomal lesions in patients. Expected differences in the signal intensity in regions affected by copy number polymorphisms were detected and, consequently, if found in MDS patients were deemed not significant. However, 4 of 33 (12%) control individuals showed isolated areas of UPD affecting a relatively small portion of 10q21.21 (2.35 Mb), 6q14.3q15 (2.36 Mb), 11q22.3 (2.88 Mb) or 13q31.3-q32.1 (4.69 Mb), respectively. As nonclonal lymphocytes were not available, the somatic, clonal nature of these defects was not established. The age of individuals in whom these lesions were found did not vary from age of the remaining group. Similarly, when controls were divided into two age groups, older than 44 years and 44 years and younger (median of the group: 44 years), there were no differences in the distribution of the lesions. No lesions were identified in two patients included as hematologic controls with a normal karyotype. SNP-A–based cytogenetic analysis in MDS We hypothesized that, because of the higher resolution of SNP-A, chromosomal lesions can be found in a higher proportion of patients than by using MC. In general, 50K SNP array hybridization confirmed the lesions identified by MC, but also detected new, previously cryptic, defects (Fig. 1), in particular microdeletions. Examples include defects seen in 3p14.2, 8q24.23, and 14q11.2. These lesions were confirmed using a higher resolution 250K SNP-A (data not shown). When results of SNP-A were displayed graphically, either as a genome scan (Fig. 1A) or as a single chromosome (Fig. 1B), deviations from normal DNA copy number were readily apparent. For example, SNP-A analysis confirmed the presence of monosomy 7, as seen in a patient with a 45,XY,-7 karyotype. When SNP-A assessment of heterozygosity was paired with DNA copy number analysis, more subtle DNA variants could be identified including UPD, e.g., on 7q, 11q (Fig. 1C, D). Copy-neutral LOH found by SNP-A was present, despite having two copies of each respective chromosome. This segmental copy-neutral LOH remains undetectable by traditional cytogenetic techniques, as it does not alter the chromosome banding pattern. Results of confirmatory MS genotyping and TaqMan PCR studies provided independent validation of the SNP-A findings (Fig. 1B, C, D). In total, lesions detected by SNP-A, which were not found by MC, were confirmed by MS TaqMan PCR-based quantitation using DNA from nonclonal CD3-positive cells in seven patients. The distinction between LOH resulting from deletions and that due to acquired UPD is apparent when comparing the SNP analysis for patient nos. 57 and 58. The segmental LOH on 7q in patient no. 58 occurs in the presence of a diploid

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

A

1731

#57 Whole Genome

Whole Genome ChrX

Chr7

B

C

#57 Chromosome 7

#58 Chromosome 7

CN LOH D7S1842

D7S1842 1

D7S2471

0.5

CD3+

D

0.48

Bone marrow

1

RQ

RQ

1

0.5

1.16

1

D7S2471 Normal PB

Bone marrow

#60 Chromosome 11 CN LOH D11S968 1

0.92

CD3+

Bone marrow

RQ

1 0.5

Figure 1. Detection and confirmation of SNP-A results in patients with myelodysplastic syndromes (MDS). (A) Whole genome scan from an exemplary patient. Each blue dot on the histogram represents the log2 intensity ratio for each SNP locus. Ratios are mapped horizontally in order of respective chromosomal location, corresponding to the short arm of chromosome 1 (left) through the long arm of chromosome X (right); Y chromosome DNA is not represented on the SNP-A chip. The histogram shows two areas of DNA copy reduction corresponding to chromosomes 7 and X, respectively, indicating that only one copy of each chromosome is present. The SNP-A findings are concordant with metaphase cytogenetics (MC) analysis, which showed a 45,XY,-7 karyotype. Array-generated karyograms of exemplary deletion of chromosome 7 (B), uniparental disomy (UPD) 7 (C), and UPD 11 (D). In the upper histogram of each example, red dots represent the log2 intensity ratio for each SNP locus and the blue line below it shows the averaged log2 values. The corresponding chromosome ideogram and location of heterozygous SNP calls (small green vertical bars) are also shown. Pink and blue bars below the ideogram indicate areas of loss of heterozygosity (LOH), with the thicker the blue bar the higher the probability of LOH. LOH was confirmed by microsatellite (MS) genotyping and TaqMan Real-Time PCR. The position of the MS marker selected for confirmation of LOH is shown on each ideogram (black rectangles). MS marker IDs are displayed for both genotyping and copy number. Copy number is presented as blue bars corresponding to relative quantification (RQ) scale, where 1 indicates a diploid chromosome number and corresponds to a value quantitated by TaqMan PCR with universal TaqMan probe designed to match CA repeats. The corresponding GTG-banded chromosomes are shown. Patient no. 57 (left): ideogram of chromosome 7 is shown. Decrease in copy number (CN) corresponds to LOH as demonstrated by the lack of heterozygous calls. By MS analysis two alleles in CD3þ cells but only one allele in bone marrow cells is present. Haploid copy number was confirmed using TaqMan PCR. Patient no. 58, showed contiguous areas of LOH corresponding to acquired segmental UPD in 7q, LOH was also confirmed by MS genotyping: there is one allele in bone marrow cells. Analysis not shown for CD3þ cells due to lack of sample from the same patient. Normal (diploid) copy number identified with the SNP array was confirmed using TaqMan PCR.

chromosome copy number shown both by SNP-A and TaqMan PCR relative DNA quantitation in bone marrow cells and peripheral blood, whereas the LOH occurring in chromosome 7 in patient no. 57 is associated with loss of a copy of the whole chromosome in marrow cells as compared to CD3þ lymphocytes (Fig. 1B).

When whole blood and bone marrow were analyzed for six MDS patients, SNP-A analysis produced concordant results in six of six patients, with two patients showing abnormal karyotypes (þ7q33 in one and þ8p23, þ2p22 in other). In addition, del 5q31.1 was detected in granulocytes in a patient for whom a marrow specimen was not available.

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

1732

A

Metaphase cytogenetics

50K SNP array

All patients MDS

Normal

MDS/MPD

Abnormal Non informative UPD UPD+gain/loss

MDS

B

50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0%

50% 45% 40% 35% 30% 25%

Patients in %

20% 15% 10% 5% 0%

+8

-5/5q-

20q-

-7

13q-

7q-

others

+8

-5/5q- 20q-

-7

4p+

13q-

7q-

others

Lesions

C

P<0.001

70%

Metaphase cytogenetics 50K SNP

60%

Number of lesions

Metaphase cyto. No Growth 0

50%

P=0.002

40%

1 P<0.001

30%

Patients in %

D

20%

2

10%

3

0% 0-1

2-3

≥4

Number of lesions

4 7

50K SNP array ≥3 Non-Informative 0 ≥1 Non-Informative 0 1 ≥2 Non-Informative 1 ≥3 3 ≥4 6 6

No. of Patients 4 1 9 21 1 1 7 17 1 1 5 1 1 1 1

Figure 2. Frequency and types of lesions detected by SNP-A as compared to traditional MC. (A) Comparison of the frequency of normal, abnormal, and noninformative studies in MC analysis vs SNP-A analysis for all patients MDS and MDS/MPD; upper portion) and MDS only (lower portion). The insert in the SNP-A pie on the right shows the portion of abnormalities attributed to acquired UPD, either as sole changes or in addition to other abnormalities. (B) Distribution of various cytogenetic abnormalities as a percentage of all abnormalities detected in MC when normal karyotypes were excluded (left), and distribution of the same abnormalities as detected by SNP-A analysis expressed as a percentage of all abnormalities detected by SNP-A (right). (C) Percentage of patients with 0 to 1, 2 to 3, and 4 or more lesions, respectively, as detected by MC and SNP-A. (D) How SNP-A results relate to the findings of MC. Of note is that for the few samples that produced noninformative results by SNP-A repeated attempts did not yield a sufficient call rate likely to due to the poor quality of DNA that was available for these patients.

Increased precision of SNP-A–based karyotyping Fewer patients had normal karyotypes by SNP-A as compared to MC (14% [10 of 72] vs 43% [31 of 72]; p ! 0.001). In 68% (21 of 31) of patients with a normal MC,

new chromosomal lesions were identified (Fig. 2A). In 80% (4 of 5) of patients in whom MC was unsuccessful, SNP-A analysis identified abnormalities. Moreover, additional lesions were found in 81% (29 of 36) of patients with

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

previously known defects (Fig. 2B, C, D). Using morphologic diagnosis as a gold standard, the resulting sensitivity for detection of chromosomal changes in MDS by SNP-A was 82% vs 53% for cytogenetics (Fig. 2A). In total, 14 of 54 lesions identified by cytogenetics were not found by SNP-A, while an abnormal karyotype was confirmed in 94% of patients (Fig. 2D). In contrast to hematologic controls with a nonclonal disease, when two MDS patients with pancytopenia with nondiagnostic marrow morphology were analyzed, the SNP array detected del(4)(q24-q25), upd(2)(q24.1-q24.3), and upd(10)(q21.2-q22.1) in one patient and upd(8)(q11.21-q11.23) in the other. The most frequently encountered lesions detected by SNP analysis occurred in chromosomes 8, 7, 5, and 11 (Fig. 3, Fig. 4) with 16, 9, 8, and 6 lesions, respectively (see Table 2 for the list of unique and shared lesions and their locations). Moreover, SNP-A showed a higher proportion of patients with chromosomal defects ($1 lesion; 82% [59 of 72] vs 50% [36 of 72]; p ! 0.001 and $2 lesions; 54% [39 of 72] vs 14% [10 of 72]; p ! 0.001) for SNP-A and MC, respectively). For example, additional previously cryptic lesions were found in patients in whom a solitary lesion found by MC was also confirmed by SNP-A evaluation. Based on the hypothetical assumption that chromosomal aberrations detected by SNP-A carry a similar prognostic

value to those detected by MC, array-based methods could result in a higher proportion of complex ($3) karyotypes (42% [30 of 72] vs 6% [4 of 72]; p ! 0.001) and consequently a higher International Prognostic Scoring System score in 50% of patients. High frequency of UPD Segmental LOH due to acquired UPD was found in 24 of 72 (33%) MDS patients by SNP-A analysis. LOH was distributed across all World Health Organization subtypes and was present both as a solitary lesion (11%), as well as in association with additional defects (22%). Acquired UPD was present in regions frequently affected by deletions as demonstrated by traditional cytogenetics, including 7q and 11q (Fig. 4). All acquired UPD lesions originated from somatic mutations, since these lesions were not found in germline DNA isolated from CD3þ lymphocytes. Clinical correlations SNP-A analysis identified new genetic abnormalities in several patients (Fig. 3). Particularly informative were lesions that seemed not to be random and/or that occurred in an overlapping fashion in multiple patients, as they point toward a unifying minimal critical region that may produce

Patient #50

Patient #23 Chr 7

1733

Chr 14

Chr 21 CN

LOH

Patient #26 Chr 5

Chr 1

Patient #57 Chr 1

Chr 11

Chr 21

Figure 3. Examples of cytogenetic and SNP array findings in selected subjects with segmental uniparental disomy. Metaphase chromosomes and SNP analysis results are shown for each example. Patient no. 23 with del(7q) revealed by both traditional cytogenetics (partial karyogram) and SNP analysis (note reduction in copy number signal on 7q) also showed UPD in the telomeric region of 14q with no visible abnormality in chromosome 14. Similar lesions are shown for patient no. 26 with del(5q) and acquired UPD in the telomeric region of the short arm of a cytogenetically normal chromosome 1, and for patient no. 57 with monosomy 7 and acquired UPD in cytogenetically normal chromosomes 1, 11, and 21. Acquired UPD was also detected in patients with normal cytogenetics; patient no. 50 with a normal karyotype showed acquired UPD in the distal long arm of chromosome 21.

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

1734

A

Chromosome 1 q-arm

p-arm

Pt # 61 Pt # 26 Pt # 57 Pt # 38 Pt # 20 Pt # 55 Pt # 59 Pt # 1 Pt # 2 Pt # 46 Pt # 16 Pt # 46 Pt # 32 Pt # 25

Chromosome 5 p-arm

Loss Gain UPD

Chromosome 11 p-arm

q-arm

q-arm

Pt # 14 Pt # 46 Pt # 26 Pt # 44 Pt # 22 Pt # 49 Pt # 48 Pt # 59

Chromosome 8

Chromosome 7

p-arm

p-arm

q-arm Pt # 4 Pt # 10 Pt # 16 Pt # 47 Pt # 48 Pt # 20 Pt # 53 Pt # 59 Pt # 7 Pt # 64 Pt # 28 Pt # 14 Pt # 61 Pt # 8 Pt # 50 Pt # 57

B

Pt # 60 Pt # 57 Pt # 51 Pt # 28 Pt # 42 Pt # 53

q-arm Pt # 66 Pt # 27 Pt # 57 Pt # 23 Pt # 63 Pt # 58 Pt # 18 Pt # 5 Pt # 64

1.0 Normal MC chr7 abnormalities by SNP-A

0.9 0.8 0.7 0.6 0.5 0.4 0.3 survival

0.2

p=0.047

0.1 0

5

10

15

20

25

30

35

40

45

months

Figure 4. Summary of frequently occurring abnormalities detected in SNP-A by chromosome and survival of patients with chromosome 7 abnormalities. (A) Chromosomes 1, 5, 7, 8, and 11 were most frequently found to have abnormalities by SNP-A analysis. Blue, green, and red bars demonstrate regions of loss, gain, or acquired UPD, respectively. While deletions involving chromosomes 5q and 7q, and trisomy 8 are common findings in this subject cohort, the SNP array revealed unexpected segmental uniparental disomy involving 1p, 1q, 8p, 8q, 7q, and 11q. (B) Demonstrates Kaplan-Meier analysis of patient cohorts with normal MC and chromosome 7 abnormalities as detected by SNP-A. Black rectangles represent censored data.

a particular phenotype. As expected, certain chromosomes were more often affected, including loss of chromosomal material within chromosomes 1, 5, 7, and 11, or gain of chromosome 8 (Fig. 4A).

In order to answer the question about clinical relevance of the newly detected lesions, we studied survival of patients with previously cryptic abnormalities of chromosome 7 (including UPD). Patients with chromosome 7 lesions

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

1735

Table 2. Chromosomal lesions detected by single nucleotide polymorphisms in patients with myelodysplastic syndromes 1

2

3

4

5

gain(p31.1) gain(p31.1)

gain(p12) gain(p12)

gain(p14.2) gain(p26.3)

gain(p15.2) gain(p15.2)

gain(p) gain(q12.1-pter)

gain(p31.1)

gain(p12)

gain(p26.3)

gain(p15.2)

del(q)

gain(p31.1)

gain(p12)

UPD(p22.3-p24.1)

gain(p15.2)

del(q11.2-q12.1)

gain(p31.1) gain(p21) gain(p31.2-p35.3) gain(p22.3) gain(p32.2)

gain(q37.3)

gain(q) gain(q31.2)

gain(q37.3) gain(q37.3)

UPD(p)

gain(q37.3)

UPD (p35.1-pter) UPD (q24.2-25.3) UPD (q25.2-25.3) UPD (q32.1-41) del(p31.1) del (p35.3-p36.31)

gain(q37.3)

UPD(q12.3-q13.11) UPD (q26.31-q26.31) del(p24.2)

gain(p15.2) gain (p15.33-p16.1) gain (p15.33-p16.1) del(q13.11-q22.1) gain(p16.1) del(q13.13-q25.32) gain(q22.1) UPD (q21.22-q22.3) del(q24)

del(q12.1-qter) del(q14.3-q23.2) del(q14.3-q34)

6

7

gain(p25.3) gain(q27)

gain(p21.3) UPD (q21.11-qter) UPD(p12.1-q12) UPD (q31.31-q31.33) UPD(p21.1-pter) UPD (q31.32-qter) UPD(q12-q13) -7 UPD -7 (q14.1-q14.2) del(q27) -7

del(q21.1-q31.3) del(q21.1-q33.2)

del(q21.11) del (q21.11-q32.3)

8 þ8 þ8 þ8 þ8 þ8 þ8 þ8 þ8 gain(p11.21-p12)

del(q21.1-q34)

gain(q24.3)

del(q23.1-q34)

gain(q37.3)

gain (q24.23-q24.3) UPD(p22-p23.2)

UPD(p22.3)

UPD(q23.1-q23.3)

UPD(p24.1-p24.3)

del(p12-pter)

UPD(p24.2-p24.3) UPD(q21.1-q22.2)

del(q24.23) del(q24.23)

del(p13.1-q11.2) del(q36.3) 9 gain(q21.11) gain(p21.1-pter)

17 gain(q22) gain(12) UPD (q11.2-qter)

10 UPD (p11.21-q11.22) del(q23.1)

18 gain(q12.2)

11 gain(p12)

12 þ12

13 þ13

14 gain(q12)

UPD(q13.4-q22.1) UPD(q13.5-q14.3) UPD(q13.5-qter) del(p12-p14.1) del(q23.3)

del(p12.33)

gain(q21.32) UPD(q31.1-q31.2) del(q12.3-q21.33) del(q13.3-q21.31)

gain(q24.1) UPD(q24.2-qter) del(q11.2) del(q11.2) del(q11.2) del(q11.2) del(q11.2) del(q12)

19 gain(p13.3) gain(p13.3)

20 gain(p12.1) gain(p12.1)

gain (q13.32-q13.33)

gain(p12.1)

21 22 þ21 UPD (q22.11-q22.2) UPD (q22.11-q22.3) del(q22.13-q22.3)

gain(p13-q13.13) gain(q11.21-pter) gain(q13.33) UPD(q13.2-q13.32) del(q11.21-q13.2) del(q11.21-q13.32) del(q11.21-q13.32) del(q11.22-q13.2) del(q12-q13.31) del(q13.2-q13.33)

15 16 del(q21.1-q22.1) del(q) del(q)

X (Y) del(q27.1-q27.2)

Significant chromosomal defects were listed by chromosome. Common/shared lesions in three or more patients were identified 1: gain(p31.1) n 5 5; 2: gain(p12) n 5 4; 2: gain(q37.3) n 5 6; 4: gain(p15.2) n 5 5; 5: del(q23.1-31.3) n 5 7; 7: UPD(q31.31-31.33) n 5 3; 8: gain(p11.21-12) n 5 9; 8: gain(q24.3) n 5 10; 11: UPD/del(q13.5-14.3) n 5 4; 14: del(q11.2) n 5 5; 19: gain(p13.3) n 5 3; 20: gain(p12.1) n 5 3; 20: del(q11.21-13.2) n 5 7; 21: UPD/del(q22.11-22.3) n 5 3.

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detected by SNP-A showed impaired survival in comparison to those with normal karyotype (p 5 0.047) (Fig. 4B). There were three patients with UPD within chromosome 7, of whom two had normal MC. Of note is that the third patient had a germline gain of Y. In general, survival of patients with SNP-A–detected defects did not differ from the survival of patients with classical 7/7q deletions by MC (median survival: 18 months in both groups). When we compared SNP-A results within low-risk MDS group with unilineage (RA, RARS) and multilineage (RCMD, RCMD-RS) dysplasia an abnormal karyotype was detected in 93% (14 of 15) and 84% (16 of 19), respectively. UPD was present in 47% (7 of 15) of patients with RA/RARS vs 26% (5 of 19) with RCMD/RCMD-RS. One case of RCMD was not informative due to poor call rate. Certain defects were identified in multiple patients. For example, gain of chromosome 4 was present in 4 of 18 patients with ringed sideroblasts; in 2 of these patients, an identical 4p15 lesion was found, while the other 2 showed an identical 4p15.33-p16.1 change. Other interesting defects not previously described in the context of MDS included changes on chromosome 1p31.1 (n 5 6), add(2) (q37) (n 5 6), and del(14)(q11) (n 5 5). Some of the lesions (e.g., add(8)(q24), del(3)(q13)] detected by SNP-A have been described in MDS in the Cancer Genome Anatomy Project (available at: http://cgap.nci.nih.gov). As a potential global measure of chromosomal instability, we also examined the total number of lesions per patient, irrespective of their chromosome location. As compared to MC, more patients showed multiple defects (Fig. 2C), which, as expected, were associated with more advanced disease. Clearly, identification of several defects in a patient corresponds to a more complex phenotype, as each of the lesions may modify the clinical behavior of the dysplastic clone.

Discussion In MDS, chromosomal aberrations have many clinical implications [3,4], but a significant proportion of patients do not exhibit cytogenetic abnormalities, while in some patients, cytogenetic analysis may not provide an informative result. High-density SNP-A offer the potential for detecting cytogenetically cryptic unbalanced chromosome rearrangements with an increasingly precise level of resolution. Previously, we have reported the first application of this technology in MDS [16], and this study describes the results of analysis of a large cohort of patients with various subtypes of MDS, as well as many technical aspects of SNP karyotyping. Our results show that SNP-A analysis of DNA isolated from dysplastic marrow allows for detection of known and previously cryptic lesions. This increased detection rate is due to the improved resolution of the genomic

scan as compared with traditional cytogenetics, while its sensitivity remains comparable. As a result, at the resolution of 50K SNP-A, the proportion of patients with a normal karyotype decreased compared to routine MC, and more patients showed multiple lesions. It is likely that the presence of additional changes as detected by SNP-A analysis is responsible for the variability of the clinical phenotype associated with a known karyotype. Analysis of these new defects indicates that they tend to occur in genomic areas that are frequently involved in known nonrandom chromosome changes. Of significant interest is that regions of UPD detected by SNP-A were also located in portions of chromosomes frequently affected by genomic losses. SNP-A constitutes a major advance in comparison to MS-based identification of LOH. Theoretically, resolution of SNP-A is limited only by the number of SNP probes used, while its sensitivity depends on the contribution of the dysplastic clone to the overall cellular content of the sample. One major advantage of SNP-A is the ability to perform analyses on interphase cells without the need for cell division. This advantage is reflected by the low proportion of noninformative results. Unlike traditional MC, SNPAs do not allow for the distinction of multiple clones, and minor clones may remain undetected due the ‘‘dilution’’ effect. The clonal nature of the lesions, including microdeletions and duplications, was confirmed using MS analysis and independent array analysis using 250K SNP microchips. In general, SNP-A showed very good correlation with MC and was confirmed by quantitative MS analysis. However, in a proportion of patients, lesions detected by MC were not found by SNP-A. This discrepancy is due to various reasons, including the fact that gain/loss of Y (n 5 4) is not tested on the array and balanced translocations and inversions [n 5 3; inv9(p11q12), t(7;8), t(1;6)] cannot be detected by this technique. Finally, the remaining discrepancy is due to lower sensitivity of SNP-A for smaller clones (n 5 7; þ9 [9 of 20], þ8 [4 of 20], and þ11 [2 of 20]). Our analysis was also performed using in a subcohort of patients’ total bone marrow and blood. So far, our results show good concordance, suggesting that, in the future, blood SNP-A may supplant analysis of bone marrow in some conditions. In analogy to whole blood, granulocytes from a patient with a known 5q-syndrome showed del5q by SNP-A. However, it is possible that in some types of MDS, mature myeloid cells are derived from residual normal stem cells precluding detection of clonal defects [22]. The content of nonclonal lymphocytes in the sample may affect the sensitivity of SNP-A. Despite this shortcoming, clonal lesions were detected in a large proportion of patients, suggesting that the clones detected were of significant size. While this clearly decreases the sensitivity, it may be an advantage, as only large, likely clinically significant clones were studied. In case of deletions, MS analysis allowed for approximation of the contamination by nonclonal cells.

L.P. Gondek et al./ Experimental Hematology 35 (2007) 1728–1738

Identification of a high frequency of segmental LOH due to UPD is a new finding that has significant implications for the pathogenesis of MDS and it extends the previous observation of a high frequency of LOH in chromosome 1 using MS scanning [23]. Importantly, LOH can also occur as a result of mitotic recombination, during which segments of homologous chromosomes are exchanged, leading to UPD [8,9], a feature that is not recognizable in MC because the chromosome banding pattern remains preserved. This mechanism of LOH has been described in many solid tumors [6,7,24,25], but has been observed rarely in myeloid malignancies [26,27]. One exception is the UPD9p associated with w30% of patients with polycythemia vera [10]. Previously, we have shown that other chromosomal regions can be affected by copy-neutral loss of heterozygosity in MDS [16]. Applicability of SNP-A to study UPD has been also demonstrated in myeloproliferative syndrome as a disease model [19], but in myeloproliferative syndrome/ MDS in particular cooperating areas of UPD (other than UPD9p) may be present. Of note is that, in our study, the somatic nature of UPD was determined by comparison with nonclonal lymphocytes. In controls, we were not able to confirm the clonal nature of UPD due to lack cells with germ line configuration. Deletions of chromosomal material could represent ‘‘unrepaired’’ lesions while defects ‘‘repaired’’ by mitotic recombination using the homologous chromosome would generate UPD and those lesions corrected using a nonhomologous chromosome could lead to translocations. We have shown that the presence of acquired segmental UPD in MDS is due to a somatic event; these results were independently confirmed by MS genotyping and PCR quantitation of locus copy number. SNP-A technology is undergoing constant improvement, with higher-density arrays being introduced at a rapid rate. While initially cost-prohibitive, wide application of SNP-A and introduction of multiple commercial platforms contributes to significant decrease in costs (around $250 with reagents and labor) that may allow for the possibility of a diagnostic application of this method in conjunction with routine MC. However, before routine clinical application, clinical correlations have to be established, a significant task for the future that can require significant time frames and is now complicated in MDS by availability of several therapies that modify the outcomes. The diversity of lesions and/or lesion combinations may also add to the complexity of such an analysis. Apart from the clinical phenotype conveyed by the individual/multiple defects detected by SNPs, their sheer numbers may be revealing as to the potential underlying pathogenesis of MDS, and points toward a global defect in the DNA machinery. In the future, SNP-A–based karyotyping may complement metaphase karyotyping in improved prediction of clinical phenotype and prognosis the diagnosis of MDS and allow for better mapping of the

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breakpoints. Our results also underscore the importance of conventional cytogenetics in clinical practice as the gold standard, because the current resolution of SNP-A technique may not detect cytogenetic abnormalities present in a smaller proportion of cells.

Acknowledgments This work was supported by National Institutes of Health grants R01 HL082983 (J.P.M.), U54 RR019391 (J.P.M.), K24 HL077522 (J.P.M.) and a charitable donation from Robert Duggan Cancer Research Foundation.

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