Leukemia (1998) 12, 1591–1595  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Malignant myeloid transformation with isochromosome 7q in Shwachman–Diamond syndrome Y Dror1, J Squire2, P Durie3 and MH Freedman1

1Division of Hematology/Oncology and 3Division of Gastroenterology and Nutrition, Department of Paediatrics, The Hospital for Sick Children and University of Toronto; and 2Ontario Cancer Institute, Toronto, affiliated with the University of Toronto, Toronto, Ontario, Canada

Shwachman–Diamond syndrome is an autosomal recessive boy underwent routine bone marrow aspiration and biopsy disorder characterized by exocrine pancreatic dysfunction, with cytogenetic analysis as part of a surveillance program to bony metaphyseal dysostosis, various degrees of cytopenia, and a striking tendency to develop myelodysplastic syndrome detect occult or early malignant myeloid transformation. Bone and acute myeloblastic leukemia. Isochromosome 7 [i(7q)] is a marrow testing revealed hypoplasia with decreased myeloid rare non-random cytogenetic abnormality of myeloid cells in precursors. No dysplastic morphologic changes were hematological malignancy. We report two cases of Shwach- detected. Blasts with a malignant appearance constituted 5% man–Diamond syndrome in which patients developed myelo- of the nucleated cells. Because of the cytogenetic abnormali- dysplastic syndrome and i(7q), detected by G-banding karyo- ties found (see Results) he is currently awaiting bone marrow type analysis and fluorescence in situ hybridization. Three transplant from a matched unrelated donor. This case has other children have been previously reported to have myelo- 2 dysplastic syndrome in association with i(7q); two of them been previously reported. had Shwachman–Diamond syndrome. Isochromosome 7q may be a fairly specific marker of myeloid malignant transformation in this syndrome and play a role in its pathogenesis. Case 2 Keywords: Shwachman–Diamond syndrome; myelodysplastic syndrome; isochromosome 7q A 4-month-old boy presented with steatorrhea and intermit- tent neutropenia. Pancreatic stimulation test results were abnormal, and a diagnosis of SDS was made. He had no evi- Introduction dence of metaphyseal dysostosis on skeletal survey. Bone marrow examination at diagnosis showed a hypoplastic speci- Shwachman–Diamond syndrome (SDS) is a multisystemic men, a relative decrease in the number of mature myeloid autosomal recessive disorder characterized by exocrine pan- forms, and prominent mast cells. The boy has not had severe creatic dysfunction, bony metaphyseal dysostosis, and various bacterial infections but requires supplemental pancreatic hematological manifestations. These include varying degrees enzymes. At the age of 5 years, as part of the surveillance of cytopenia and a marked tendency in up to one-third of program, he underwent routine bone marrow aspiration and patients to develop myelodysplastic syndrome (MDS) and biopsy, which again showed a hypoplastic specimen. The 1 acute myeloblastic leukemia. Isochromosome 7q [i(7q)] is a myeloid precursors were decreased in number with dysplastic rare cytogenetic abnormality of bone marrow myeloid cells changes. Blasts constituted 5% of the nucleated cells. Mast associated with hematological malignancy. We report two cells were again prominent. Because of the presence of the cases of SDS complicated by MDS and i(7q) and relate them cytogenetic abnormalities described (see Results) he is also to two earlier reported cases of a similar abnormality. awaiting bone marrow transplant from a matched unrelated donor. Case reports Methods Case 1 G-banding analysis A 10-month-old boy presented with steatorrhea due to exo- crine pancreatic insufficiency, severe intermittent neutropenia Freshly obtained bone marrow cells collected in preservative- and moderate thrombocytopenia. Pancreatic stimulation test free heparin were analyzed for cytogenetic abnormalities. results were abnormal, and a diagnosis of SDS was made. Both direct (uncultured) cells and 24-h cultures were then har- Additional findings included reactive airway disease, mildly vested using standard protocols.3 were analyzed elevated hepatic transaminases, right supernumerary thumb using a conventional GTG-banding method. ISCN 1995 and severe dental caries. The boy had not suffered from severe criteria were used to define abnormal clones.4 infectious complications. He was treated with oral pancreatic enzymes and fat-soluble vitamins. Cytogenetic analysis of peripheral lymphocytes was normal. Bone marrow examin- Fluorescence in situ hybridization (FISH) analysis ation, performed at diagnosis, showed a hypoplastic specimen with decreased myelocytic precursors. Cytogenetic analysis of Slides made from the bone marrow samples were prewarmed marrow cells was not performed. At the age of 6.5 years the in 2 × SSC (1 × SSC = 0.15 M NaCl/0.15 M sodium citrate) pH 7.0 at 37°C for 30 min. The slides were then dehydrated in ethanol and denatured in a solution of 70% formamide in 2 × Correspondence: Y Dror, Division of Hematology/Oncology, The ° 5 Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, SSC at 70 C, as previously described. Slides were hybridized Canada M5G 1X8; Fax: 416 813 5327 to commercially available probes at 37°C in a humidified Received 19 March 1998; accepted 22 June 1998 chamber overnight. A DNA probe specific to locus D7S522 Shwachman syndrome with isochromosome 7q Y Dror et al 1592 at 7q31 directly labeled with spectrum orange LSI D75522 ino-2-phenylindole (DAPI), the slides were evaluated on an (Vysis, Downers Grove, IL, USA) and centromer spectrum epi-fluorescence microscope for interphase scoring. The green D7Z1 (Vysis) were used. The slides were then washed hybridization signals were enumerated in 200 interphase with 0.4 × SSC, 0.3% NP-40 solution at 72°C and with 2 × nuclei for each specimen. Nuclei with an additional 7q signal SSC, 0.1% NP-40. After being counterstained with 4,6-diam- were scored as positive for the presence of i(7q).

a

b

Figure 1 (a) G-banding from patient 1 with i(7q) indicated by an arrow. (b) FISH interphase cell from patient 1. The centromer of is indicated by a green fluorescence and the long arm by an orange fluorescence. The upper part of the nucleus containing two orange signals confirms the presence of the isochromosome. Shwachman syndrome with isochromosome 7q Y Dror et al 1593 a

b

Figure 2 (a) G-banding analysis from patient 2 with i(7q) indicated by an arrow. The abnormal clone containing del(20)(q11) is not shown. (b) FISH cell from patient 2. I(7q), identified by chromosome-specific paint, is placed centrally in the metaphase spread. The second normal chromosome 7 is seen at the upper left field. Shwachman syndrome with isochromosome 7q Y Dror et al 1594 painting was performed using whole chromosome painting tial portion of patients with SDS who develop MDS/acute probe for chromosome 7 (WCP7; Oncor, Gaithersburg, MD, myeloid leukemia will also exhibit chromosome 7 abnormali- USA) to identify 7q abnormalities on metaphase preparations ties. In this report we describe two children with SDS who from patient 2. acquired I(7q). I(7q) has been reported only in three children with myelodysplasia;1,9,10 and interestingly two of them also had SDS.1,9 The third child developed pancytopenia and Results hypoplastic bone marrow at age 5 years but did not have pan- G-banding analysis creatic insufficiency, although his height and weight were below the 5th percentile for age.10 This rare structural aber- For patient 1, 7 metaphase cells were analyzed and all ration of chromosome 7 is completely different from the more showed 46,XY,i(7)(q10) (Figure 1a). For patient 2, 20 meta- common form, monosomy 7, but both seem to be related to phase cells were analyzed: two showed 46,XY,i(7)(q10) the development of malignant myeloid transformation. (Figure 2a), 11 showed 46,XY,del(20)(q11), and seven showed An isochromosome consists of two identical copies of one normal male karyotype. chromosome arm. It is caused by either abnormal splitting of the during mitosis11 or a propensity of sequences FISH analysis in one proximal arm for breakage and reunion events between sister or homologous .12 This results For patient 1, interphase FISH was performed using a 7(q)- in a loss of one arm while the other is duplicated to form one specific probe. I(7q)-positive cells were identified by observ- symmetric chromosome with two genetically identical arms. ing three signals over the nucleus, diploid cell by observing Thus an isochromosome for the long arm of number 7 – i(7q) – two signals (Figure 1b). Twenty-four positive cells were ident- contains two copies of the long arm separated by a centro- ified among 200 cells counted. For patient 2, interphase mere. This structural abnormality leads to monosomy for the analysis was inconclusive; metaphase FISH was therefore per- genes on 7p and trisomy for those on 7q. formed. I(7q)-positive cells were identified by observing two The mechanism by which chromosome 7 abnormalities similar fluorescence signals surrounding the same nucleus contribute to the pathogenesis of malignant myeloid trans- (Figure 2b). Two positive cells were identified among 14 formation is not known. Since the common forms of chromo- cells counted. some 7 abnormalities involve of 7q, and since tri- somy of this arm occurs in i(7q), disruption of proximal genes Discussion on 7q is a possible mechanism. Possible candidate genes are the human homologues of the yeast DNA mismatch repair A review of the medical literature in English revealed 14 gene PMS2 at 7q11.22–23,13 mutations of which are found in patients with SDS that transformed into MDS/acute myeloid hereditary nonpolyposis colon cancer.14 Another leukemo- leukemia with abnormal bone marrow cytogenetic studies.1,6–9 genic mechanism might be a loss of gene(s) on the short arm Nine of these patients had cytogenetic abnormalities involving of chromosome 7 such as human PMS2 gene, a replicate of chromosome 7 (Table 1). These cases suggest that a substan- which maps to 7p22.14 Alternatively, chromosome 7 abnor-

Table 1 Bone marrow cytogenetic abnormalities reported in patients with Shwachman–Diamond syndrome

Patient Chromosomal abnormalitya Subtype Ref.

1 46,XY,i(7)(q11) Refractory multi-lineage cytopenia Present study 2 46,XY/46,XY,del(20)(q11)/ Refractory multi-lineage cytopenia with myeloid Present study 46,XY,i(7)(q10) lineage dysplasia 3 47XY,+1,del(9)(q22) RAEBT → AML-M5 1 4 46XY,i(7q) RA 1 5 46XY,der(7)t(4,7)(q31:q11)+21 RA → AML-M5 1 6 46,XX,der(7)(q11.2q32) RA 1 7 46–47,XY,−2,−4,del(5)(q23q33), RAEBT → AML-M6 1 del(7)(q22)+2−3r,+2−4mars 8 46,XY,add(11)(p),−15,−22, RAEBT → AML-M2 1 +marl,+mar2 9 53,XY,+G,+G ALL-L1 6 10 45,XY,−7,mar18 Pancytopenia → MDS/AML 6 11 47,XY,+21,+4q,mar1q Pancytopenia → MDS → AML-M4 6 12 46,XY/46,XY,del(7)(q22-q34) Neutropenia → MDS 7 13 46,XX/45,XX,t(6;13)(q21-q32),−7 MDS 7 14 Inv(9) Severe aplastic anemia → MDS/AML-M4 8 15 45XY,−7/46XY,I(7q) Hypoplastic bone marrow with fibrosis 9 16 45,XY,−7 MDS 9

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome without further details; RA, refractory anemia; RAEBT, refractory anemia with excess blasts in transformation. aChromosome 7 abnormalities are indicated by italics and boldface type. → indicates transformation. Shwachman syndrome with isochromosome 7q Y Dror et al 1595 malities may merely be an epiphenomenon associated with 4 Mitelman F (ed). ISCN 1995. An International System for Human myeloid malignancies but not directly involved in their patho- Cytogenetic Nomenclature. S Karger: Basel, 1995. genesis, being prone to breakage once the malignant trans- 5 Lichter P, Tang C, Call K, Hermanson G, Evans G, Housman D, Ward D. High resolution mapping of human by formation occurs. in-situ hybridization with cosmid clones. Science 1990; 247: Interestingly, Smith et al’s patient and patient 1 in the 64–69. present study had i(7q) as the sole abnormality, a finding that 6 Woods WG, Roloff JS, Lukens JN, Krivit W. The occurrence of supports the hypothesis of a mechanistic role of the chromo- leukemia in patients with the Shwachman syndrome. J Pediatr some 7 abnormality in the multistep pathogenesis of the 1981; 99: 425–428. malignant transformation. It is noteworthy that our patient 2 7 Kalra R, Dale D, Freedman MH, Bonilla MA, Weinblatt M, Ganser A et al. Monosomy 7 and activating RAS mutations accompany exhibited i(7q) as a minor clone; the majority of the abnormal malignant transformation in patients with congenital neutropenia. cells showed del(20)(q11), another chromosomal abnormality Blood 1995; 86: 4579–4586. reported in MDS.15 8 Arsenier L, Dietrich H, Link H. Allogeneic bone marrow transplan- Chromosome 7 abnormalities are common in a variety of tation in a patient with Schwachman–Diamond syndrome. Ann conditions with a propensity for MDS, including congenital Hematol 1996; 72: 83–84. bone marrow disorders such as SDS and Kostmann’s syn- 9 Okcu F, Roberts WM, Chan KW. Bone marrow transplantation in Schwachman–Diamond syndrome: report of two cases and review drome and acquired conditions such as severe aplastic ane- of the literature. Bone Marrow Transplant 1998; 21: 849–851. 16 mia. The broad spectrum of underlying predispositions asso- 10 Murray JC, Mahoney DH Jr, Cooley LD. Isochromosome 7q in ciated with chromosome 7 abnormalities suggests that these childhood myelodysplastic syndrome. Leukemia 1996; 10: 746– aberrations are secondary events that contribute to the devel- 747. opment of leukemia. The genetic defect and molecular basis 11 De la Chapelle A. How do human isochromosomes arise? Cancer for SDS that predisposes patients to changes in chromosome Genet Cytogenet 1982; 5: 173–179. 12 Wolff DJ, Miller AP, Van Dyke DL, Schwartz S, Willard HF. Mol- 7, and the genes on chromosome 7 responsible for the malig- ecular definition of breakpoints associated with human Xq iso- nant transformation, require definition. chromosomes: implications for mechanisms of formation. Am J Hum Genet 1996; 58: 154–160. 13 Osborne LR, Herbrick JA, Greavette T, Heng HHQ, Tsui LC, Scherer SW. PMS2-related genes flank the rearrangement break- References points associated with Williams syndrome and other diseases on human chromosome 7. Genomics 1997; 45: 402–406. 14 Nikolaides NC, Papadopoulos N, Liu B, Wei YF, Carter KC, Ruben 1 Smith OP, Hann IM, Chessells JM, Reevers BR, Milla P. Haemato- SM et al. Mutations of two PMS homologues in hereditary non- logical abnormalities in Shwachman–Diamond syndrome. Br J polyposis colon cancer. Nature 1994; 371: 75–80. Haematol 1996; 94: 279–284. 15 Third MIC Cooperative Group Recommendations for a morpho- 2 Dror Y, Durie P, Marcon P, Freedman MH. Duplication of distal logic, immunologic, and cytogenetic (MIC) working classification thumb phalanx in Shwachman–Diamond syndrome. Am J Med of the primary and therapy-related myelodysplastic disorders. Can- Genet 1998; 78: 67–69. cer Genet Cytogenet 1988; 32: 1–10. 3 Le Beau MM. Cytogenetic analysis of hematological malignant dis- 16 Luna-Fineman S, Shannon KM, Lange BJ. Childhood monosomy eases. In: Barch MA (ed). The ACT Cytogenetic Laboratory Man- 7: epidemiology, biology, and mechanistic implication. Blood ual, 2nd edn. Raven Press: New York, 1991, pp 396–445. 1995; 85: 1985–1999.