Clonal evolution in marrows of patients with Shwachman-Diamond syndrome

Experimental Hematology 30 (2002) 659–669

Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: A prospective 5-year follow-up study Yigal Drora, Peter Durieb, Hedy Ginzbergb, Rebecca Hermana, Anu Banerjeec, Martin Champagned, Kevin Shannonc, David Malkina, and Melvin H. Freedmana Department of Paediatrics, Divisions of aHematology and Oncology and bGastroenterology and Nutrition, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada; cThe University of California at San Francisco, San Francisco, Calif., USA; d Hôpital Ste.-Justine, Montréal, Québec, Canada (Received 22 February 2002; revised 19 March 2002; accepted 20 March 2002)

Objectives. Shwachman-Diamond syndrome (SDS) is characterized by varying degrees of marrow failure. Retrospective studies suggested a high propensity for malignant myeloid transformation into myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The study’s aims were to determine the cellular and molecular characteristics as well as the clinical course of malignant myeloid transformation and clonal marrow disease in patients with SDS. Methods. This is a longitudinal prospective study of 14 patients recruited for annual hematological evaluations. Results of baseline and serial hematological assessments for up to 5 years are reported. Results. Clonal marrow cytogenetic abnormalities (CMCA) were detected in 4 patients (29%) on first testing or at follow-up. The abnormalities were del(20q) in two patients, i(7q) in one, and combined del(20q) and i(7q) in one. The following tests did not distinguish patients with CMCA from other SDS patients: severity of peripheral cytopenia, fetal hemoglobin levels, percentage of marrow CD34 cells, colony growth from marrow CD34 cells, cluster-to-colony ratio, marrow stromal function, percentage of marrow apoptosis cells, and granulocyte colony-stimulating factor receptor expression. RAS and p53 mutation analysis and AML blast colony assays were uniformly negative. No patients showed progression into more advanced stages of MDS or into AML. In one patient, the abnormal clone became undetectable after 2 years of follow-up. Conclusions. We conclude that although CMCA in SDS is high, progression into advanced stages of MDS or to overt AML may be slow and difficult to predict. Treatment should be cautious since some abnormal clones can regress. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Shwachman-Diamond syndrome (SDS) is an inherited multisystem disorder characterized by exocrine pancreatic dysfunction, varying degrees of cytopenia, and metaphyseal dysplasia [1–5]. Studies of family pedigrees support an autosomal recessive mode of inheritance [5]. The SDS locus has recently been mapped to the centromeric region of chromosome 7 (7p12-7q11) [6]. However, the molecular basis for the pleiotropic phenotype, the multisystem features, the Offprint requests to: Yigal Dror, M.D., Division of Haematology/Oncology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada; E-mail: [email protected]

bone marrow failure, and the leukemic transformation is still unclear. We have previously shown that bone marrow from patients with SDS is characterized by decreased frequency of CD34 cells, and that marrow CD34 cells have a reduced ability to form hematopoietic colonies in vitro [7]. SDS patients also have a serious generalized marrow dysfunction with abnormal marrow stroma in terms of its ability to support and maintain normal hematopoiesis. The central pathogenetic mechanism of the bone marrow failure in SDS appears mediated by increased apoptosis [8]. This increased propensity for apoptosis is linked to increased expression of

0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(02)0 0 8 1 5 - 9

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the Fas antigen and hyperactivation of the Fas signaling pathway [8]. The development of malignant myeloid transformation, including various myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), has been described in 8 to 33% of patients [3,5,9]. Since all the series reported have been retrospective [3,9]or cross-sectional [5], the precise incidence and cumulative risk of malignant myeloid transformation are unknown. The present study is the first report of a long-term prospective study designed to determine the cellular and molecular characteristics as well as the clinical course of malignant myeloid transformation and a clonal marrow cytogenetic abnormality (CMCA) in patients with SDS. This report describes the results of the initial and serial assessments of 14 SDS patients for up to 5 years. Because the prognosis of patients who develop advanced MDS or AML is dismal [2,3,10,11], our goal is to identify risk factors and markers of malignancy in an early phase before advanced clinical transformation occurs.

Materials and methods Patient population and clinical diagnosis Patients with an established diagnosis of SDS were recruited since January 1997. Patients were included if they fulfilled our predetermined diagnostic criteria for SDS [5,6]. Those already known to have malignant myeloid transformation were excluded. All the studies described were approved by our hospital’s Research Ethics Board. Informed consent was obtained from the patients or their parents for drawing an additional 3–5 mL of bone marrow for research purposes. Control samples were obtained from 12 hematologically normal, healthy donors for bone marrow transplantation; informed, written consent was obtained in all cases. Twelve patients have been previously reported [4,5,7,8,12,13]. Criteria for the diagnosis of malignant myeloid transformation Malignant myeloid transformation was diagnosed if a patient developed one of the following: prominent morphologic dysplasia, increased percentage of marrow blasts (5%), or the appearance of a clonal cytogenetic abnormality. Stage of MDS was defined according to the Category-Cytology-Cytogenetics (CCC) classification of childhood MDS (Table 1) [14,15]. To remedy the lack of a useful classification system for pediatric MDS, we recently developed the CCC system, which incorporates the “category” of MDS (de novo, syndromic, or therapy-related), the “cytology” (refractory cytopenia, refractory cytopenia with dysplasia [RCD], refractory cytopenia with ring sideroblasts [RCRS], and refractory cytopenia with excess blasts [RCEB]), and the “cytogenetics.” Complete blood count and fetal hemoglobin Peripheral blood was drawn for complete blood counts, mean corpuscular volume, and reticulocyte count. Fetal hemoglobin concentrations were determined by high-pressure liquid chromatography [16].

Table 1. The “CCC” classification of pediatric myelodysplastic syndromes Category idiopathic/de novo syndrome-related treatment/toxin-related Cytology refractory single/multilineage cytopenia with ring sideroblasts (RCRS) refractory single/multilineage cytopenia without obvious dysplasia (RC) refractory single/multilineage cytopenia with dysplasia (RCD) refractory single/multilineage cytopenia with excess blasts (5–30%) (RCEB) Cytogenetics Positive (CG) Negative (CG) Unknown (CG0) From [14,15]. Used with permission of Lippincott Williams & Wilkins.

Bone marrow samples Patients underwent bone marrow aspiration and biopsy from the posterior superior iliac crest following sedation with intravenous propofol and local infiltration with lidocaine. Morphological examination of the bone marrow Bone marrow aspirates were spread directly on slides, stained with Wright-Giemsa, and examined independently by our institutional hematopathologist and by YD and MHF. Bone marrow core biopsies were obtained with a Jamshidi needle, placed in formalin, embedded in paraffin, and stained with hematoxylin and eosin. Morphological findings of dysplasia were described as previously proposed [17]. Determination of MDS subclass was based on our proposed CCC classification for pediatric MDS [14,15]. Bone marrow cytogenetics Bone marrow cells collected in preservative-free heparin were analyzed for cytogenetic abnormalities. Both direct (uncultured) cells and 24-hour cultures were then harvested using standard protocols, and metaphases were analyzed via a conventional GTG-banding method [18] Abnormal clones were defined according to the 1995 International System for Human Cytogenetic Nomenclature (ISCN) criteria [19]. Fluorescent in-situ hybridization (FISH) analysis using case-specific chromosome markers was performed if abnormality was detected by standard cytogenetics. Percentage of CD34 cells in marrow cell samples Bone marrow aspirates collected in preservative-free heparin underwent Ficoll (Pharmacia Biotec, Uppsala, Sweden) fractionation. Light-density cell layer was collected and washed in phosphate-buffered saline/bovine serum albumin 0.5%. Cells (1  105) were incubated as previously described [7] with either fluorescein isothiocyanate (FITC)-conjugated anti-CD34 antibody or IgG1FITC isotype control (both from Immunex, Seattle, WA, USA), resuspended in phosphate-buffered saline with paraformaldehyde 0.5%, and immediately analyzed by a Coulter Epics XL-MCL flow cytometer (Coulter, Hialeah, FL, USA). At least 12,000 events were collected. Short-term clonogenic assays Bone marrow aspirates collected in preservative-free heparin underwent Percoll fractionation. Light-density mononuclear cells underwent CD34 enrichment by the Mini-MACS immunomagnetic separation system (Miltenyi Biotec, Auburn, CA, USA) as previously

Y. Dror et al./Experimental Hematology 30 (2002) 659–669

described [8]. CD34 cells were plated in duplicate at a density of 3  103 cells/mL in culture wells containing methylcellulose (Fluka, Buchs, Switzerland), 40 U/mL interleukin-3 (Immunex, Seattle, WA, USA), 10 ng/mL granulocyte colony-stimulating factor (G-CSF; Amgen, Thousand Oaks, CA, USA), 50 ng/mL mast cell growth factor (Immunex), and 2 U/mL erythropoietin (Ortho Biologics, Manati, PR). Cultures were incubated for 14 days, then scored for colony (50 cells) formation: granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), or mixed colonyforming units (CFU-mix). Marrow mononuclear cells were plated at a density of 2  105 cells/mL in similar conditions and scored after 14 days for cluster (groups of 20–50 cells) to colony (50 cells) ratio. For megakaryocyte colony-forming unit (CFU-Meg) assays, 0.2  105 CD34 cells were placed in 0.2-mL cultures (in a 24well plate) containing methylcellulose, 50 ng/mL mast cell growth factor (Immunex), and 50 ng/mL megakaryocyte growth and development factor (Amgen). Colonies of at least 3 mature megakaryocytes were scored after 14 days.

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leukin-3 (1  102 U/mL), interleukin-6 (2  102 ng/mL), interleukin-11 (10 U/mL), granulocyte-macrophage colony-stimulating factor (35 U/mL), and stem cell factor (5 ng/mL). Cultures were incubated, then observed after 7 and 14 days under an inverted microscope for the development of colonies that contained a pure population of greater than 20 cells with the morphologic appearance of leukemic blasts. Long-term marrow stromal cell cultures Stromal layers from 10 patients and 11 normal subjects were established as previously described [7] by plating 2  106 post-Percoll bone marrow mononuclear cells in 2 mL long-term culture (LTC) medium (Myelocult, Stem Cell Technologies, Vancouver, BC, Canada), containing 12.5% fetal calf serum, 12.5% horse serum, 0.1 mmol/L 2-mercaptoethanol, 105 mol/L hydrocortisone, 0.016 mmol/L folic acid, 2.0 mmol/L glutamine, and 0.16 mmol/L inositol. LTC were incubated in a 33C incubator for 4–5 weeks, and stromal layers were fed weekly by one-half medium replacement. Support of hematopoiesis was then assessed as previously described [7] by crossover experiments performed by plating 2  105 control CD34 cells over patient or control stromal layers after irradiation (15 Gy). Nonadherent cells (6  103), harvested weekly

AML blast colony assay Post-Percoll marrow mononuclear cells (1  105 cells/mL) from 9 patients were plated in methylcellulose cultures containing inter-

Table 2. Hematological characteristics of patients with Schwachman-Diamond syndrome at presentation and follow-up Bone marrow morphology UPN

Year

Age (y)

1

1997 2001 1997

7 12 5

2001

10

1997 2000 1998 2001 1997 2001 1997 2001 1997 1999 1997 2001 1997 2001* 1997 2001* 1997 2001 1999 2001 1999 2001 2001

14 17 16 19 13 16 2 5 14 17 17 22 6 10 1 5.5 7 11 16 18 5 6 1.5

2

3 4 5 6 7 8 9 10 11 14 15 16

Sex M M

M F F M F M F F M M F F

Blood counts

MCV FL

HBF (%)

Cell

N,A,T N,A,T N,A

90 88 86

9.7 6.5 9.4

⇓ ⇓ ⇓

.⇓ ⇓ d,l

N,A

85

9

n

N N N,T N,T N,A N N,A N N,A,T N,A,T N,A,T N,A,T N,A N,T N,A,T A,T N,T N,T N,A,T N N N N,A

93 91 101 100 93 92 82 78 92 93 102 104 82 80 79 76 93 96 83 82 84 ND 91

1 1 1.1 1.3 3.1 4.1 3.6 2.3 1.7 1.8 2.0 2.3 4.9 ND 9.6 ND 1.4 1.4 1 1 3.l ND 9.3

⇓ ⇓ ⇓ ⇓ ⇓ n ⇑ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ US ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ND n

G

E

Meg

Blasts

Cytogenetics

n n n

⇓ ⇓ n

5% 5% 5%

n

md

n

5%

⇓ ⇓ ⇓ ⇓ 1 n ⇓md,l ⇓,md ⇓ ⇓ ⇓ ⇓,md n n n ⇓,l ⇓ ⇓ ⇓,l ⇓,l ⇓,l ND n

n n n n md n ⇑,n md n n n n n n n n n n md n n ND n

⇓ ν ⇓ ⇓ n n n n ⇓ ν ⇓ ⇓ ⇓ US US ⇓ n md n n n ND md

5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% 5% ND 5%

46,XY,i(7q)[7] 46,XY,i(7q)[3]/46,XY[47] 46,XY,del(20q)[11]/ 46,XY,i(7q)[2]/46,XY[7] 46,XY,del(20q)[19]/ 46,XY,i(7q)[1]/46,XY[5] 46,XY[13] 46,XY[19] 46,XX[11] 46,XX[81]/46,XX,del(20)(q12)[5] 46,XX 46,XX 46,XY 46,XY 46,XX US 46,XY 46,XY 46,XX 46,XX 46,XX 46,XX 46,XY 46,XY 46,XY[24]/46,XY,del(20)(q12)[1] 46,XY 46,XX ND 46,XX

Clone size (FISH, %)

CCC Subclass

12 6 14

RC/CG RC/CG RCD/CG

D

RC/CG

ND ND ND 5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10 0 ND ND ND

RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG0 RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG RC/CG TC/CG RC/CG RC/CG RC/CG RC/CG0 RC/CG

A, anemia (hemoglobin concentration 2 SD below mean, adjusted for age61; Cell, cellularity; d, dysplastic; E, erythrocyte count; F, female; HbF, fetal hemoglobin; l, left shift; Meg, megakaryocyte count; G, granulocytic lineage cell count; M, male; MCV, mean corpuscular volume; md, mild dysplasia; N, neutropenia (neutrophil concentration 1.5  109/L); n, normal; ND, not determined; T, thrombocytopenia (platelet count 150  109/L); ND, not determinecd; UPN, unique patient number; US, unsuccessful; y, year; ⇓, decreased; ⇑, increased. *Last bone marrow test was done in 1998.

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during half-medium replacement of LTC medium, were plated in 0.6 mL short-term cultures and assayed for CFU-GM as described above for short-term cultures from fresh CD34 cells. Percentage of apoptotic marrow mononuclear cells Post-Percoll light-density marrow cells were plated at a cell density of 2  105 cells/mL in 1.0-mL dishes containing the same ingredients and under the same conditions as above (see Short-term clonogenic assays) [8]. After 7 days in culture, cells were collected after dissolving the methylcellulose by incubation in the culture dishes with Iscove’s medium. To a 1  105 cell mixture, FITCconjugated annexin V (10 L, R&D Systems, Minneapolis, MN, USA) and propidium iodide (10 L, R&D Systems) were added, and cells were incubated in the dark for 15 minutes at room temperature. Subsequently, binding buffer (R&D Systems) was added, and cells were immediately analyzed by Coulter Epics XL-MCL flow cytometer (Coulter). The events falling outside the negative staining regions identified by control samples were considered positive staining for either annexin V only (apoptosis) or annexin V and propidium iodide (late apoptosis/necrotic cells). Fas expression on marrow cell subpopulations Post-Ficoll light-density marrow cells (1  105) were incubated with either phycoerythrin (PE)-conjugated mouse anti-human Fas IgM monoclonal antibody (clone 7C11, 8 L/105 cells) or PEconjugated IgM mouse isotype control (clone GC323, 8 L/105 cells) (both purchased from Immunotech). Cells were then washed and analyzed by a Coulter Epics XL-MCL flow cytometer (Coulter). G-CSF receptor expression on neutrophils Post-Ficoll light-density marrow cells (5  105) were incubated in the dark for 1 hour in 4C with either PE-conjugated IgG-1 subclass, anti–G-CSF receptor monoclonal antibodies (Fluorokine, R&D Systems), or PE-conjugated streptavidin as isotype control

(both from R&D Systems). Cells were washed and analyzed by flow cytometry. NRAS, KRAS, and p53 gene mutation analyses DNA was extracted from bone marrow cells of 8 patients as previously described [20] and suspended in Tris/EDTA buffer for use in polymerase chain reaction (PCR) experiments. DNA samples were amplified in a DNA Thermocycle machine (Perkin Elmer, Norwalk, CT, USA) with reaction mixtures that included respective 3 and 5 primers, target genomic DNA, Taq polymerase (AmpliTaq; Cetus, Emeryville, CA, USA), and deoxynucleotides. For singlestrand conformational polymorphism (SSCP), 33P deoxyadenine triphosphate or 33P deoxycytosine triphosphate was incorporated into the PCR reaction mixture to label the fragments. Oligonucleotide primers described by Suzuki and colleagues [21] were used to amplify both exons of the KRAS and NRAS fragments for SSCP and primers designed by van Mansfeld and Bos [22] for allele-specific restriction enzyme digests. PCR conditions were as described previously [20,21], and SSCP was performed as recommended by Suzuki et al. [21]. Briefly, gels were prepared from a solution of acrylamide (MDE; Hydrolink, Malvern, PA, USA) and Tris:borate EDTA (TBE). Amplified RAS fragments were diluted in a solution of NaOH, EDTA, and deionized formamide, then denatured at 95C before loading. Gels were poured in, plated, and electrophoresed at 8 W of constant power at both room temperature and 4C for 16 hours, followed by autoradiography. Exons 2 through 11 of the p53 gene were PCR amplified from 50 ng of DNA as previously described [23] Exons 2 and 3 were amplified together, allowing determination of the status of intron 2. SSCP analysis of the PCR products was then performed. The PCR product of each exon was examined under 2 to 4 gel conditions with varying glycerol and acrylamide content. Direct mutation analysis of a separate aliquot of DNA was performed on any exonic fragments that had demonstrated band shift by SSCP.

Table 3. Studies of marrow cells from patients with Schwachman-Diamong syndrome and normal control subjects Pts with CMCA (UPN)* Test Marrow CD34 cells (%) Clonogenic assays CFU-GM† BFU-E CFU-mix† CFU-Meg† Cluster/colony ratio Long-term cultures Nonadherent cells‡ Fas expression (%) Apoptotic cells§ (%) GCSFR expression (%)

Pts without CMCA (n 10)

Controls (n 11)

1

2

4

14

Mean

SEM

Range

Mean

SEM

Range

2.3

ND

1.4

ND

2.1

0.8

0.2–4.9

5.5

0.8

2.2–10

0 0 0 4 1.8

26 40 7 16 ND

2 12 0 4 0.3

92 120 10 ND 0.2

24 24 2 22 0.5

6.6 7.3 0.5 13.7 0.1

0–72 0–74 0–5 0–136 0.2–0.7

79 52 11 81 0.16

14.6 20.5 2.2 29.2 0.03

30–130 13–137 5–20 27–180 0–0.4

0.14 ND ND 46

ND 28 33 63

ND ND ND ND

0.16 28 22 45

0–0.85 16–46 10–38 27–84

1.4 13 14 67

0.47 1.1 2.9 4

0.03–3.89 5–19 3–27 31–85

0 28 43 68

0.09 5.6 4 5

Abbreviations: CMCA, clonal marrow cytogenetic abnormality; Pt, patient; ND, not determined; CFU-GM, granulocyte-monocyte colony-forming units; BFU-E, erythrocyte burst-forming units; CFU-mix, mixed colony-forming units; CFU-Meg, megakaryocyte colony-forming units; SEM, standard deviation of the mean. *Tests were performed at the time the cytogenetic abnormalities were present. † Difference between means for patients without CMCA and normal controls was significant (p  0.05). ‡ Harvested at week 6. § Apoptosis of marrow mononuclear cells after 7 days incubation in clonogenic assays.

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Data analysis Unpaired two-tailed Student’s t-test was used to determine the statistical significance of differences in colony numbers in short-term cultures, numbers of nonadherent cells harvested weekly from the long-term cultures, levels of G-CSF receptor and Fas expression, and percentage of apoptotic cells between patient and control samples. p values of less than 0.05 were considered significant.

Results Patient population and clinical diagnosis Of 16 patients recruited into the study, 2 were excluded because they did not fulfill our diagnostic criteria. The remaining 14 included 7 males and 7 females; their ages at entry to the study ranged from 1 to 18 years (mean 9 years, median 7.5). The clinical characteristics of the patients are shown in Table 2. All the patients showed evidence of pancreatic dysfunction, and 9 were pancreatic-insufficient and required pancreatic enzyme supplements with meals and snacks. No patient was transfusion-dependent or had been treated with hematopoietic cytokines. CMCA was detected in 3 patients on first testing and in an additional patient after a 3-year follow-up (crude rate, 29%; see below, “Bone marrow cytogenetics”). It is noteworthy that the brother of the latter patient (UPN4) also had SDS, and died of AML 10 years before initiation of the current study. According to the CCC classification of childhood MDS, 3 patients were diagnosed as having syndromic MDS/refractory cytopenia/positive cytogenetics (syndromic MDS/RC/CG) and 1 patient had syndromic MDS/refractory cytopenia with dysplasia/positive cytogenetics (syndromic MDS/RCD/CG). Complete blood count and fetal hemoglobin Hematological abnormalities are shown in Table 2. All patients had chronic (persistent or intermittent) neutropenia ( 1.5  109/L), 10 had low hemoglobin for age, and 7 had thrombocytopenia ( 150  109/L). Five patients had trilineage cytopenia. There was no correlation between the severity of peripheral cytopenia and the identification of a CMCA. High fetal hemoglobin levels were found both in SDS patients with CMCA and in those without it. Morphological examination of the bone marrow As shown in Table 2, moderately severe single-lineage dysplastic changes were found in one child (UPN2), with giant myelocytes and metamyelocytes and coarse granulation. Mild dysplastic erythropoiesis or megakaryopoiesis was noted in several other patients. These conditions were attributed to the inherent nature of the bone marrow failure and disordered hematopoiesis, rather than to the development of malignant myeloid transformation. None of the latter patients had a clonal marrow cytogenetic abnormality. Mild dysplastic changes can be transient, as demonstrated in 4 patients in

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our series. No patient had blast counts of more than 5% of nucleated marrow cells. Bone marrow cytogenetics Table 2 shows that all patients had informative cytogenetic analysis of bone marrow cells on each marrow testing except for one follow-up test in UPN7. Evidence of a clonal cytogenetic abnormality was found in 4 children (29%) at ages of 5 to 19 years; 3 were male and 1 was female. Abnormalities included del(20)(q12) in 2 patients, i(7)(q10) in 1, and combined i(7)(q10) and del(20q) in 1. FISH analysis was performed in all cases (interphase and metaphase). Percentage of the abnormal clones fluctuated and ranged from 5 to 43% (Table 2).[TBL 2] Percentage of CD34 cells in marrow cell samples As shown in Table 3, percentage of CD34 cells in marrows from 8 SDS patients (mean SEM: 1.8% 0.5%; median: 1.4%) was significantly lower (p  0.01) than the percentage from 10 normal controls (mean SEM: 4.4% 0.5%, median: 5.4%). However, the percentage of CD34 cells in marrows from 2 patients with CMCA who were assessed (1.4% and 2.3%) did not differ from the others. Standard clonogenic assays When the clonogenic potential of patients’ marrow CD34 cells was examined, no difference was found between the patients with and without CMCA (Table 3), although mean values of CFU-GM, CFU-mix, and CFU-Meg colonies were lower than those of normal controls (p  0.05). Mean BFU-E colony numbers were also lower (26 vs 52), but the difference was not significant (p 0.14). When bone marrow mononuclear cells were plated in clonogenic assays, the ratio of cluster-to-colony formation from UPN1 (1.8) was much higher than those of the 7 other patients without CMCA who were studied (mean, 0.61; range, 0.19–0.74), which is characteristic of MDS in evolution [24]. However, in 2 other patients with CMCA, the ratio was in the range of the other SDS patients without CMCA. It is noteworthy that the SDS patients as a group had significantly higher (p  0.05) cluster-to-colony ratios (mean, 0.61; range, 0.19–1.77) than normal controls (mean, 0.10; range, 0.03–0.18). AML blast colony assays We attempted to expand the abnormal myelodysplastic clones from 2 patients with CMCA as well as from the 7 other patients with SDS but without CMCA. However, no pure colonies containing more than 20 blast cells were identified at days 7 and 14. Long-term marrow stromal cell cultures Plating normal CD34 cells over SDS stromal layers resulted in lower numbers of nonadherent cells at weeks 3–6 (p  .05) than in long-term cultures in which normal

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CD34 cells were plated over normal stroma (Table 2). However, when 2 patients with CMCA were evaluated, their stromal function in terms of nonadherent cell production from normal CD34 cells was indistinguishable from the patients without CMCA (Table 3). The stromal cultures were not controlled for HLA type. However, no differences were observed between autologous and allogeneic combinations, in keeping with previous observations by us [7] and others [25]. Percentage of apoptotic marrow mononuclear cells As shown in Table 3, bone marrow mononuclear cells plated in methylcellulose cultures showed an increased percentage of apoptotic cells (mean SEM, 37% 3 vs 13% 2 in controls, p  0.05). Interestingly, patient UPN1, who had CMCA, also had a high proportion of cells undergoing apoptosis (43%), exceeding the range of the patients with SDS without CMCA (mean 24.1%, range 10–38%). However, in another patient with CMCA, the results were only in the high range of the other SDS patients. Fas expression on marrow cells As shown in Table 3, Fas expression on post-Ficoll marrow cells from the SDS patients was significantly higher than in normal controls (mean SEM, 28% 4 vs 13% 2, p  0.05). However, its expression in the 2 patients with CMCA did not differ from the values obtained from SDS patients without CMCA. G-CSF receptor expression on neutrophils G-CSF receptor expression on bone marrow granulocytes was quantified by flow cytometry (Table 3). UPN1 (68%), UPN2 (46%), and 6 non-MDS patients (mean, 54%) had receptor expression levels comparable with those of normal control subjects (mean, 67%; p 0.37). RAS and P53 gene mutation analyses No mutations in codons 12 and 13 of KRAS and NRAS were identified in 2 patients with CMCA and in 7 other SDS patients without CMCA. Nor were mutations identified in the regions of p53 examined in 2 cases of CMCA and in an additional 9 cases. Follow-up studies During the 5 years of the study, 3 patients were found on first testing to have marrow cytogenetic abnormalities. The levels of the abnormal clones did not increase on subsequent testing; in one patient (UPN1), the level decreased from 14% to 6%, and in another (UPN14) it disappeared on standard cytogenetic and FISH analyses after 2 years of follow-up. The third patient (UPN2) has relatively stable clones by standard karyotype testing, but did not have repeat FISH analysis. The fourth patient (UPN4) had normal cytogenetic analyses in 2 successive years but developed an abnormal clone in the third year. No patient in the series de-

veloped excessive blasts (RCEB or leukemia) during the period of the study. There was no prominent, consistent change in the severity of the cytopenia throughout the study in any of the SDS patients (with or without CMCA). Fetal hemoglobin levels in the patients with CMCA either slightly decreased (in UPN1 from 10.7 to 6.5%) or stayed at the same levels. The other patients without CMCA showed no significant changes in fetal hemoglobin levels.

Discussion This is the first report of a prospective study designed to characterize the clinical and laboratory evolution of patients with malignant myeloid transformation and CMCA in patients with SDS. The findings at presentation and at 5 years of follow-up are reported. CMCA without increased percentage of blasts or overt leukemia was discovered in 4 of 14 SDS patients (29%). None of them had symptoms related to low blood counts or required transfusions. Thus, malignant myeloid transformation was disclosed at a relatively early phase: syndromic MDS/RC/CG or syndromic MDS/ RCD/CG, according to the CCC classification. However, in children [15,26] as in adults [27], these stages of MDS frequently evolve into more advanced stages of MDS and AML. It is noteworthy that clonal or oligoclonal hematopoiesis may be present without detectable cytogenetic abnormality; thus, patients without CMCA may also have clonal or oligoclonal hematopoiesis. A major question is the significance of clonal cytogenetic abnormalities in marrows of patients with SDS. Of the patients with MDS and AML, 75% [15,26] and 84% [28] have CMCA, respectively. Clonal disease is one of the most important prognostic factors in childhood MDS [15,26], and most patients deteriorate and progress into advanced AML or MDS [15,26,27]. Further, deletion of the long arm of chromosome 20, as was found in 3 of the patients in the present series, is a well-known abnormality observed in patients with MDS and AML [29]. Review of the literature revealed that seven reported SDS cases with cytogenetic abnormalities occurred without prominent marrow dysplasia or increase in blasts (RC/CG) (Table 4). Of these patients, 2 had stable disease, 2 developed morphological dysplasia or followed a more severe clinical course, 1 developed AML, and 2 could not be evaluated. Therefore, the finding of a clonal cytogenetic abnormality in morphologically “benign” bone marrow aspirates is likely of clinical significance, and requires frequent monitoring. We thus view CMCA as a marker of malignant myeloid transformation. Since CMCA is only one of multiple events occurring during leukemogenesis, the malignant myeloid transformation in such cases can be incomplete or complete, reversible or irreversible. Cytogenetic abnormalities have been reported in 24 SDS patients in various stages of malignant myeloid transfor-

Y. Dror et al./Experimental Hematology 30 (2002) 659–669

mation: RC/CG, RCD, RCEB, and AML (Table 4) [4,9,10,26,30–39]. We were the first to notice an increased incidence of isochromosome 7q [i(7q)] in SDS [4]. Six of the patients so far reported (22%) had i(7q), an extremely uncommon cytogenetic abnormality, described rarely in MDS, AML, or acute lymphoblastic leukemia patients without SDS [40–42]. The high percentage of i(7q) in SDS strongly suggests that it is a fairly specific clonal marker in this syndrome and may even be related to the SDS gene, which is located on the centromeric region of chromosome 7 (7p12-7q11) [6]. The possible relationship between the SDS gene and the i(7q) formation, which also involves breakpoints at 7q(10-11), may be elucidated when the gene is identified, cloned, and sequenced. Other chromosome 7 abnormalities have been described in an additional 10 patients, including complete monosomy 7 in 7 patients (one had combined i[7q] and monosomy 7) and deletion of part of the long arm of chromosome 7 in 2 patients (Table 4). These cases suggest that a large proportion of patients with SDS with malignant myeloid transformation acquire chromosome 7 abnormalities. However, complete monosomy 7 or partial deletions of its long arm are also characteristic of patients with other inherited bone marrow failure syndromes who develop MDS or AML, including Fanconi’s anemia, Kostmann’s neutropenia, and congenital amegakaryocytic thrombocytopenia [40]. Other cytogenetic abnormalities have been described in SDS at various disease stages (Table 4).] The prognostic significance of the various cytogenetic abnormalities in SDS still has to be deciphered. It is noteworthy that among the 6 reported patients with i(7q) no progression into RCEB or AML has been described. In contrast, 4 of the patients with the other chromosome 7 abnormalities either presented with or progressed to RCEB (1) or AML (3) from earlier stages of MDS. The abnormality i(7q) has also been associated with morphological dysplasia in one patient, transfusion dependency in another, and previous G-CSF therapy for severe neutropenia in a third. These data may signify a different prognostic value of i(7q) in this disorder. A primary goal of this study was early detection of MDS before it progressed to an advanced and possibly fatal disease [26]. MDS in SDS also has a dismal prognosis [3,9,11], resulting in death either from bleeding, from infectious complications, or from conversion to AML [3,9,11, 33,34]. Currently, there are no prospective follow-up data on the outcome of patients diagnosed at such an early stage as our patients with CMCA, and the rate of progression remains unclear. Most of the previously reported patients with SDS and abnormal marrow cytogenetics who can be evaluated for progression had progressive disease, and some had stable disease (Table 4) [2,9,40]. The present study showed for the first time that in SDS the abnormal clone can regress and becomes undetectable by conventional methods. This phenomenon has been described in other rare conditions

665

[43,44], but not in SDS and not for del(20q). This finding has important implications for planning the treatment of patients with SDS. The only available cure for patients with SDS who develop MDS is hematopoietic stem cell transplantation. However, high transplant-related mortality occurs in cases of SDS with advanced MDS or AML [10,30,33,34]. For these reasons—and since, as we found, an abnormal clone in an SDS marrow can regress—we now recommend close monitoring of such patients, monthly complete blood counts, and bone marrow examinations initially every 3 months. Signs of progression that will prompt immediate consideration of a transplant include recurrent infection, platelet or red blood cell requirement, and elevated marrow blast counts. In order to characterize the dysplastic nature and leukemogenic events of marrows from SDS patients and specifically those with CMCA, we screened marrow specimens from SDS with or without CMCA with various tests known to be abnormal in MDS. Although several findings unique to MDS/AML were found characteristic also to SDS, no obvious differences were found between patients with CMCA or without. There was no correlation between severity of peripheral cytopenia and development of CMCA. It is noteworthy that one of the patients with CMCA had prominent morphological dysplasia (UPN2), and an additional patient had increased percentage of apoptotic cells (UPN1). In addition, 11 patients had fetal hemoglobin levels exceeding our age-adjusted reference values (0.1–1.1%) for children older than 1 year. Three patients had much higher levels than the others, and 2 of them had CMCA. However, the other 2 patients with CMCA had normal or only slightly elevated levels. Since elevation of fetal hemoglobin (10%) is considered a poor prognostic factor in childhood MDS [26], monitoring its levels is recommended. Whether fetal hemoglobin level has prognostic value in SDS in terms of developing severe aplasia, advanced MDS, or leukemia remains to be determined by longer prospective monitoring. Apart from significant dysgranulopoiesis in Patient UPN2, all patients with an abnormal marrow karyotype showed the same bone marrow characteristics as the other SDS patients, which included negative periodic acid-Schiff stain, absence of ring sideroblasts, and low blast counts. Mild dysplastic changes were commonly noted and were discounted as a feature of MDS. In addition, in agreement with previous reports [45,46], some morphologically dysplastic changes may be transient also in SDS. This was documented in 4 patients in the present series. Compared to normal controls, bone marrows of SDS patients showed lower numbers of CD34 and reduced in vitro hematopoietic colony formation potential of CD34 cells [7]. Similar to MDS [47], SDS is also characterized by abnormal stromal function [7]. However, no difference was noted among patients with or without MDS with regard to these marrow parameters. Interestingly, the cluster-to–colony formation ratio in clonogenic assays from patient UPN1

Table cases of Shwachman-Diamond with clonal marrow cytogenetic abnormality 666 4. Transformation stage and outcome of 25 Y. reported Dror et al./Experimental Hematology 30syndrome (2002) 659–669 Age at BMA/ No Gender

Marrow Cytogenetics

CCC Diagnosis*

Therapy



Outcome

Follow-up Post Therapy

1

7/M

46,XY,i(7)(q11)

RC/CG

—

SD

4y

2

5/M

RC/CG

—

SD

4y

3

9/M 10 11 2/M 3.5/M 4.5 11/F 42/M

RC/CG RCEB/CG AML-M5/CG RCD/CG RCD/CG AML-M5/CG RCD/CG RCEB/CG

— Supportive Supportive — — Supportive NM Chemotherapy

PD PD PD* PD PD PD* PD NR

— — * Alive at 2 y — * Alive —

AML-M6/CG

NM

8/M 8 1.5/M

46,XY/46,XY,20(q11)/ 46,XY,i(7)(q10) XY,1,9(q22) 47,XY,,9(q22) 47,XY,1,9(q22) 46,XY,i(7q) 46,XY,der(7),t(4,7)(q31;q11) 47,XY,der(7),t(4,7)(q31;q11),21 46,XY/46,XX,der(7)(q11.2;q32) 46,XY,del(20q)/46–47,XY,2,4, 5(q23;q33),7(q22)2–3r, 2–4mars 46,XY,20q/46–47,XY,2,4, 5(q23–q33),7(q22)2–3r,2–4mars 46,XY,11p,15,22,marl,mar2 46,XY,11p,15,22,marl,mar3 53,XY,G,G

RAEB/CG AML-M2/CG ALL-L1

Chemotherapy NM Chemotherapy

Pulmonary aspergilosis* NR Sepsis* Alive, well

10

9/M

45,XY,7,mar18

AML/CG

Chemotherapy

11

NM/F 2.6

NM 47,XY,21,4q,marlq

RCD/CG AML-M4/CG

Chemotherapy NM

46,XY,7(q22–q34) 46,XY,7(q22–q34) 45,XX,t(6;13)(q21–q32),7 Inv(9) Inv(9) 45,XY,7

RC/CG RCD/CG RCD/CG RCD/CG AML-M4/CG RCEB/CG

NM NM NM

15

13/M NM 14/F 23/M 24 8/M

16

11/M

45,XY,7/46,XY,i(7q)

RC/CG

17

38/M

45–50,XY,18,t(21;?)(q22;?), dic(22;?)(p11;?) 45–50,XY,18,t(21;?)(q22;?), dic(22;?)(p11;?)

RCEB/CG

Mismatched related donorHSCT —

AML-M4/CG

Chemotherapy, Alive, well MUDHSCT

AML-M2/CG

Chemotherapy

CR, alive

RC/CG RCD/CG RCD/CG

— MRD-HSCT MUD-HSCT

RC/CG

MUD-HSCT MUD-HSCT None then supportive

Alive CR CR, no major complications Respiratory failure* Graft failure* PD, severe aplasia*

4 5 6 7

43 8 9

12 13 14

38

18

17/M

19 20 21

10/F 5/F 13/M

22

8/F

49–52,XY,X,5,8,8,9,12p, 13,marl-4(cp12) 45,XX,-C(FISH 45,XX,7[99%]) inv(14)(q11q32) 46XY,7(q22–q34)/ 46XY,7(q23–q34),21 46XX,i(7q)

23 24

7.5/F 5/F

45XX,t(6:;13)(q21;q32),7 46XX,i(7q10)RC/CG

RCD/CG RC/CG

25 26

16/F 16/M

46,XX,20(q12) 46,XY,20(q12)

RC/CG RC/CG

— MRD-HSCT MUD-HSCT

— —

Severe aplasia, sepsis* Severe aplasia Pulmonary hemorrhage* NM NM NM PD Relapse* CNS-, GVHD-3, renal acidosis, hypoglycemia, pneumonitis* CNS-4, GVHD-4, pulmonary hemorrhage*

Reference 4, UPN1, present paper 4, UPN2, present paper 9, 26

9 9 9, 26 9, 30

* — * 12m (post diagnosis) *Several weeks —

9, 26 11 11 11,31

*3m NM NM NM

32

— * *93d Post-HSCT

*31d post-HSCT

PD

32 33 10

10

—

34

97d post-HSCT (36 months, personal communication) 2y

35

1y 2y 12m

36 37 38

*2m

38

*2m *10y

38 39

SD 4y, Alive Disappearance of 2.5y clone (RC/CG)

UPN4, present paper UPN14, present pape

F, female; HSCT, hematopoietic stem cell transplantation; M, male; m, month; MRD, matched related donor; MUD, matched unrelated donor; NM, not mentioned; NR, no response; PD, progressive disease; SD, stable disease; UPN, unique patient number; y, year. *CCC diagnosis, see text.

Y. Dror et al./Experimental Hematology 30 (2002) 659–669

was much higher than in 7 non-MDS patients. This reflects “preleukemic” hematopoiesis in this patient [24]. It is noteworthy that the other SDS patients without CMCA had significantly higher cluster-to-colony ratios than observed in clonogenic assays from normal controls. This finding appears consistent with the preleukemic nature of the syndrome. Similar to what occurs in patients with adult-type MDS [48,49], marrow cells from patients with SDS have an increased tendency to undergo apoptosis [8] and increased Fas expression [8]. However, there was no evidence of progression among the patients with CMCA. Only one patient with CMCA (UPN1) had more apoptotic cells than the other SDS patients without CMCA. Neutropenia is the most common hematological abnormality in patients with SDS. In Kostmann’s neutropenia, another form of congenital neutropenia, there is a similar propensity to develop malignant myeloid transformation, which is usually associated with an acquired mutation of the G-CSF receptor on myeloid cells [50]. In addition, an abnormal expression of G-CSF receptor was noted on the blast cells from patients with de novo AML [51,52]. For these reasons we measured G-CSF receptors on neutrophils at the protein level. Patients UPN1 and UPN2 and the patients without CMCA all had normal expression of the receptor. Therefore, neutropenia and the development of CMCA in SDS do not appear to be associated with quantitative changes in expression of the G-CSF receptor. Approximately 20 to 30% of the pediatric cases of MDS show RAS oncogene mutations, usually in codons 12 and 13 of KRAS and NRAS, and there is evidence that these activating mutations in RAS-mediated signal transduction are involved in leukemia-associated dysregulation in myeloid growth and differentiation [53,54]. Nevertheless, in the present study 2 of the 4 patients with SDS who had CMCA and 7 additional SDS patients without CMCA had no mutations in codons 12 and 13 of KRAS and NRAS. One case of SDS with malignant myeloid transformation in which RAS oncogene mutations had not been detected was previously reported [32]. Although these results suggest that RAS mutation may not play a role in the early evolution of MDS in SDS, prospective monitoring is needed to answer this question definitively. In adults, p53 mutations have been reported in approximately 10% of MDS cases [55,56 and in 3 to 7% of AML cases [57–59]. The frequency of p53 mutations in cases of de novo MDS/AML in children as well as among cases of malignant myeloid transformation related to inherited bone marrow failure syndromes is not known. Similar to our RAS oncogene findings, it appears that rearrangement in this tumor suppressor gene is not involved in the early stage of malignant myeloid transformation in SDS. The similarity in prevalence of p53 protein overexpression in SDS marrow samples (even without malignant myeloid trnasformation) and refractory anemia patients [60] is interesting. If this

667

finding is confirmed in larger studies using various anti-p53 antibodies, it may implicate either a posttranslational modification or an increased gene expression. The possible role of p53 protein overexpression and increased apoptosis [8] should be clarified. To conclude, our results show a high incidence (29%) of CMCA in patients with SDS. The cumulative incidence is likely to be higher because our study entailed only a 5-year assessment, and patients previously diagnosed with overt leukemia or MDS at our hospital [3] were excluded. However, progression from isolated CMCA into advanced stages of MDS or to overt AML may be slow and difficult to predict. Further, we showed herein that an abnormal clone consisting of del(20q) can be transient. Prospective evaluation of these patients over a longer period is still necessary to accurately determine the incidence of CMCA and its longterm clinical implications, which patients are at higher risk of transformation, and whether patients with an early bone marrow transplantation results in an improved long-term outcome. Acknowledgments We are indebted to Wilma Vanek and Elizabeth Sexsmith for technical assistance. This article was prepared with the assistance of Editorial Services, The Hospital for Sick Children, Toronto, Canada. The work was supported in part by grants from the Audey Stanley Memorial Leukemia Research Fund, Shwachman Diamond Support Canada, Aplastic Anemia Association of Canada, Elizabeth Rose Herman Fund, and The Severe Chronic Neutropenia International Registry.

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