Clinical and molecular cytogenetic studies in seven patients with myeloid diseases characterized by i(20q−)

Authors


Dr Yongquan Xue, The First Affiliated Hospital of Soochow University, Jiangsu Institute of Haematology, 96 Shizi Street, Suzhou 215006, China. E-mail: uujihsmc@public1.sz.js.cn

Summary

We report on seven patients with myeloid diseases characterized by i(20q−) anomaly. Four patients were male and three were female, their median age was 57 years. The diagnosis at presentation was myelodysplastic syndrome in six patients, acute myeloid leukaemia in one patient. Four died but three survived and remain anaemic. The survivals were 6 months for patient 1, 7 months for patient 2, 17 d for patient 4 and 28 d for patient 5. Chromosome specimens were prepared by direct and/or short-term culture of bone marrow cells. Karyotype analysis was performed by R- and G-banding technique, which showed that one of the normal chromosomes 20 was substituted by one or two small metacentric chromosomes in all seven patients. The karyotype was ider(20)(q10)del(20)(q11q13), i.e. i(20q−) in six patients by dual-colour fluorescence in situ hybridization assay using two probes (a subtelomeric probe for 20q and an unique probe for 20q12). As far as we know, this anomaly has not been reported previously. Thus, we consider that i(20q−) is a novel and rare recurrent chromosomal abnormality that is specifically associated with myeloid diseases and may indicate a poor prognosis.

Deletion of the long arm of chromosome 20 represents one of the most common chromosomal abnormalities associated with myeloid diseases. It is found in 10% of patients with polycythaemia vera (Reeves et al, 1972; Diez-Martin et al, 1991) as well as in other myeloproliferative disorders (Demory et al, 1988; Mertens et al, 1991). It is also seen in approximately 5% of patients with myelodysplastic syndrome (MDS) (Davis et al, 1984; Knapp et al, 1985) and 1–2% of patients with acute myeloid leukaemia (AML) (Heim & Mitelman, 1992). Since 1998, we have found seven patients characterized by an isochromosome of 20q arm with a loss of interstitial material, i.e. i(20q−). As far as we know, this anomaly has not been reported previously. Their clinical and prognostic features were definitely different from those with 20q− alone. Here, we describe their clinical and genetic findings.

Patients and methods

Patients

Of 998 MDS patients and 3073 AML patients that were examined cytogenetically in our hospital between 1985 and 2004, the i(20q−) anomaly was detected in seven patients (six with MDS, one with AML). The median age of the four male and three female patients was 57 years (range 51–74 years). Of these seven patients, five came from Jiangsu Province, one from Shandong Province and one from Anhui Province. In patient 3, haemoglobin electrophoresis showed HbA 21·5%, HbF 18·9%, but no bands of abnormal Hb and denatured Hb, and bone marrow biopsy revealed abnormal localization of immature precursors (ALIP) in the myeloid series and morbid haemopoiesis of megakaryocytes. In patient 4, immunophenotypic analysis showed that the leukaemic cells were positive for CD34 (57·6%), CD13 (37·3%) and CyMPO (44·6%). To date, four have died (patients 1, 2, 4 and 5 with a survival of 6 months, 7 months, 17 d and 28 d respectively); three are still alive with anaemia (patients 3, 6 and 7 after 1 year, 1 month and 18 d respectively) (calculated from the dates of diagnosis to 5 February 2004). Their clinical and haematological features are presented in Table I. Informed content was obtained from the patients or their relatives.

Table I.  The clinical and haematological features in seven patients with myeloid diseases and i(20q−) anomaly.
Patient no.Age (years)/sexClinical findingsPeripheral blood examinationBone marrow examinationDiagnosisTreatmentOutcome
  1. Hb, haemoglobin concentration; WBC, white blood cell count; Plt, platelet count; POX, peroxidase; RA, refractory anaemia; RAEB, refractory anaemia with excess of blasts; HA, homoharringtonine, cytosine arabinoside; MA, mitoxantrone, cytosine arabinoside; MDS, myelodysplastic syndrome; AML, acute myeloid leukaemia.

160/femaleAnaemiaHb: 5·7 g/dl
WBC: 1·8 × 109/l
Plt: 34 × 109/l
Hypercellular with 1% blasts, hypersegmentation in neutrophils, megaloblastoid change in nucleated red cells and micromegakaryocytesMDS (RA)Blood transfusion, testosterone propionate, stanozolol, retinoic acid, vitamin B6, Chinese traditional medicineNo response. Died 6 months after diagnosis
252/maleAnaemia and splenomegaly (lower edge at the level of umbilicus)Hb: 6·8 g/l
WBC: 6·8 × 109/l
Plt: 51 × 109/l
Hypercellular with 6·5% blasts, hypersegmentation and ringed nuclei in neutrophils, and megaloblastoid change in nucleated red cellsMDS (RAEB)Blood transfusion, HA regimen (one course)No response. Died 7 months after diagnosis
351/maleFever, anaemia and hepatosplenomegaly (both 8 cm below the costal margin)Hb: 5·8 g/dl
WBC: 4·0 × 109/l
Plt: 77 × 109 l
Hypercellular with 3·5% blasts, Pelger Huet anomaly, megaloblastoid changes in nucleated red cells and micromegakaryocytesMDS (RA)Blood transfusion, antibiotic therapyAlive and anaemic (diagnosed 14 February 2003)
458/femaleAnaemia, petechiae and ecchymosesHb: 8·7 g/dl
WBC: 6·1 × 109/l
Plt: 10 × 109/l
Hypercellular with 8·5% monoblasts, 25% promonocytes and 8·5% monocytes. POX: 2% intensive positive, 4% weakly positive, 94% negativeAML-M5bMA regimen (two courses)No response. Died 17 d after diagnosis
554/maleAnaemiaHb: 3·4 g/dl
WBC: 3·0 × 109/l
Plt: 287 × 109/l
Hypercellular with 9% blasts, hypersegmentation in neutrophils, megaloblastoid change in nucleated red cellsMDS (RAEB)Blood transfusion, routine anti-anaemia therapyNo response. Died 28 d after diagnosis
674/maleAnaemiaHb: 6·0 g/dl
WBC: 2·4 × 109/l
Plt: 66 × 109/l
Hypercellular with 5% blasts, hyposegmentation in neutrophils, binuclear in nucleated red cells and micromegakaryocytesMDS (RA)Blood transfusion, Andriol, retinoic acid, erythropoietinAlive and anaemic (diagnosed 25 December 2003)
751/femaleFever and anaemiaHb: 9·4 g/dl
WBC: 3·1 × 109/l
Plt: 12 × 109/l
Hypercellular with 4% blasts, nuclear abnormalities in nucleated red cells and micromegakaryocytesMDS (RA)Retinoic acid, vitamin B6, calcitriol, antibiotic therapyAlive and anaemic (diagnosed 19 January 2004)

Cytogenetic analysis

Chromosomes were prepared routinely by the direct method or short-term culture of bone marrow cells. Karyotypes were analysed on R- and G-banded metaphases. At least 20 metaphases were observed for each specimen. Patient 3 was analysed on three occasions, while the others were analysed only on one occasion. Chromosome abnormalities were described according to the International System for Human Cytogenetic Nomenclature (Mitelman, 1995).

Dual-colour fluorescence in situ hybridization (D-FISH)

Six patients (patients 2–7) were studied. Patient 1 did not undergo D-FISH examination due to the lack of specimen. A sample from a normal male with a karyotype of 46, XY was used as control.

Probes

The SpectrumGreen labelled subtelomeric probe for 20q was purchased from Qbiogene (Montreal, Canada). The locus-specific probe for chromosome 20q12 (YAC912C3) was provided by Dr Herve, Avet-Loiseau (Laboratoire de Cytogenetique Hematologique, Centre Hospitalier Universitaire de Nantes, France). Biotin and Nick translation test kits were purchased from Vysis (Downers Grove, IL, USA).

Pretreatment of the probes

The manufacturer's protocol was applied with slight modifications. Briefly, after RNase pretreatment, the chromosome preparations were denatured for 3 min at 72°C in 70% formamide/2 × saline sodium citrate (SSC), followed by dehydration in a cold ethanol series and air-dried. Then the preparations were treated with proteinase  K, dehydrated and air-dried. The locus-specific probe for 20q12 was labelled with biotin by nick translation. Three microlitre of this probe was mixed with 2 μl Cot-1TM DNA, 0·8 μl sodium acetate and 10 μl 100% ethanol. It was frozen at −80°C for 30 min, and then centrifuged at 11 372 g, 4°C for 30 min to deposit DNA. After washing with 70% ethanol and centrifuging at 11 372 g, 4°C for 15 min the DNA was dehydrated prior to the addition of the labelled subtelomeric probe for 20q. The preparation was denatured for 5 min at 75°C, then placed on ice and subsequently at 37°C for 30 min to prehybridize.

Hybridization

The probes were then applied to the target cell preparations, and incubated overnight at 37°C in a moist air chamber. After hybridization the slides were washed in 50% formamide/2 × SSC for 5 min followed by two washes at 45°C in 1·1 × SSC for 5 min each. The hybridization signals were detected using the conventional system of two layers of Avidin-Spectrum Red and one layer of biotinylated Anti-Avidin. Then the slides were counterstained with 4,6-diamidino-2-phenyl-indole.

Evaluation

The evaluation was performed using a fluorescence microscope (Leica AMRXA, Wetzlar, Germany). QFISH software (Leica Microsystems Imaging Solutions Ltd, Cambridge, UK) was used to scan and preserve the images. At least 10 metaphases were analysed for each sample.

Results

Chromosome analysis

Cytogenetic data for the seven patients are shown in Table II. Each of patients had lost one normal chromosome 20, which was replaced by one or two metacentric chromosomes that were smaller than the normal chromosome 20. Because their stains were similar to those of the long arm of chromosome 20, they were suspected to be an isoderivative of chromosome 20 with a loss of interstitial material, i.e. ider(20)del(20q) (Figs 1–3). Each of patients 4, 5 and 7 had a clone with only one ider(20)del(20q). The other four patients all had a clone with two ider(20)del(20q) that were of same size; of these, three had a clone with one ider(20)del(20q) simultaneously. The other chromosomal abnormalities detected included an isochromosome of the short arm of chromosome 6, i.e. i(6p) accompanied by two ider(20)del(20q), in some of the metaphases from patient 1, and a monosomy  7 accompanied by one or two ider(20)del(20q) in all of the metaphases from patient 2. A deletion of the long arm of the chromosome 20 was found to be sole abnormality in some metaphases of patient 4, and a polyploidy with two i(20q−) was seen in 13% of the mitoses of patient 7. No metaphases with normal karyotype were found in five patients except patients 5 and 7.

Table II.  The cytogenetic data of seven patients with myeloid diseases and i(20q−) anomaly.
Patient no.MethodNo. of metaphases analysedKaryotype
  1. BM, bone marrow; D, direct method; C24, after culture for 24 h.

  2. *Patient 3 was analysed on three separate occasions.

1BMD2946,XX, ider(20)del(20q)[6]/47,idem,+ ider(20)del(20q)[3]/47,idem,i(6p),+ ider(20)del(20q)[20]
2BMC242045, XY,−7,ider(20)del(20q)[17]/46,idem,+ ider(20)del(20q)[3]
3*BMC24 (1)2447,XY,−20,+ider(20)del(20q) × i2[24]
BMC24 (2)3447,XY,−20,+ider(20)del(20q) × i2[34]
BMC24 (3)4246,XY,ider(20)del(20q)[2]/47,idem,+ ider(20)del(20q)[40]
4BMC242046,XX,del(20q)[8]/46,XX,ider(20)del(20q)[12]
5BMC243146,XY,ider(20)del(20q)[27]/46,XY,[4]
6BMC242047,XY,−20,+ider(20)del(20q) × i2[20]
7BMC242546,XX,ider(20)del(20q)[23]/46,XX[2] (polyploidy accounted for 13% of the mitoses, of which, tetraploidy with two ider(20)del(20q) was predominated)
Figure 1.

G-banded karyotype from patient 1 showing 47,XX,i(6)(p10),−20,+ider(20)del(20q)x2. Inset shows partial R-banded karyotype with the same anomalies.

Figure 2.

R-banded karyotype from patient 4 showing 46,XX,ider(20)del(20q).

Figure 3.

R-banded karyotype from patient 3 showing 47,XY,−20,+ider(20)del(20q)x2.

D-FISH

In normal metaphases, each long arm of two chromosomes of F group size showed a green fluorescent signal at one terminal and a nearby red fluorescent signal (Fig 4), suggesting that they were normal chromosomes 20. In six patients, all or most metaphases showed one to two small chromosomes that had two symmetric green fluorescent signals at both terminals but did not have any red ones between them, confirming that these abnormal chromosomes were the isoderivative chromosomes generated by a deletion of 20q12, i.e. ider(20)(q10)del(20)(q11q13) (Figs 5 and 6). In patient 4, some of metaphases showed green and red fluorescent signals on the long arm of a small chromosome indicating a normal chromosome 20, but only a green fluorescent signal on the long arm of the other small chromosome, suggesting that the abnormality is a deletion of the long arm of chromosome 20 [del(20)(q11q13)] rather than ider(20)(q10)del(20)(q11q13) (data not shown).

Figure 4.

FISH analysis using a locus-specific probe for 20q12 and a subtelomeric probe for 20q showing red and green signals on the long arm of both normal chromosomes 20.

Figure 5.

FISH analysis of patient 4 using the same probe as in Fig 4 showing two symmetric green signals at both terminals of the ider(20)del(20q) but without any red signal between them, indicating an ider(20)(q10)del(20)(q11q13), the other chromosome with red and green signals is a normal chromosome 20.

Figure 6.

FISH analysis of patient 3 using the same probe as in Fig 4 showing the same signals as in Fig 5, indicating ider(20)(q10)del(20)(q11q13)x2 and a normal chromosome 20.

Discussion

All seven patients in this series had one to two isochromosomes that were smaller than chromosomes 20. Their stains were similar to the long arm of chromosome 20 on karyotype analyses with R-banding, so they were suspected of being derived from chromosome 20. This isoderivative chromosome showed lighter staining intensity in its centromere and darker staining intensity in both arms on R-banded preparations and the opposite pattern in staining intensity was observed on G-banded preparations. Through comparing the results of the two banding techniques, we consider that the R-banding technique is preferable to the G-banding technique for the identification of i(20q−). To ascertain its real origin and nature, we performed D-FISH using a probe for 20q12 and a subtelomeric probe for 20q. The results showed that the derivative chromosome had symmetric green fluorescent signals on its two terminals but did not have any red ones between them. This convincingly proved that the derivative chromosome was an isoderivative chromosome of the long arm of chromosome 20, resulting from a deletion of 20q12, i.e. ider(20)(q10)del(20)(q11q13). This kind of chromosome abnormality was found in only seven patients which accounted for 0·60% (6/998) of MDS patients and 0·03% (1/3073) of AML patients in our hospital. It is rather surprising that i(20q−) has never been described, although there have been many reports about 20q− anomaly including double 20q− (Ohyashiki et al, 1992) and multiple copies of dicentrics as a result of telomeric association between deleted 20q arms with loss of interstitial material (Falzetti et al, 1999). Thus, it is a novel but rare recurrent abnormality and we hope that our report will encourage more attention to this anomaly, and may stimulate the identification of others.

We believe that the mechanism which caused i(20q−) included two steps: the first step was the formation of del(20)(q11q13), i.e. 20q−. Some cells from patient 4 had 20q− as the sole anomaly. This fact proved 20q− to be a precedent pathological change. The second step was the generation of ider(20) due to an error in the mitotic process in which the chromosomes were separated horizontally instead of longitudinally; the long arm with partial deletion was then taken as a template to duplicate the another long arm with the same partial deletion. In some patients, the non-disjunction of i(20q−) in the following mitosis caused the duplication of i(20q−). Thus, we consider that the ider(20) comes from the 20q−. But this evolution is a very rare event that could require some prerequisites that are unknown at present.

20q− is a common recurring abnormality in myeloid diseases. Patients with 20q− alone were classified as a ‘good risk’ group (Greenberg et al, 1997). A number of consistent features have been described (Wattel et al, 1993). These include low-grade MDS, a low incidence of anaemia, infrequent progression to AML and prolonged survival, with a median survival of 48 months. The size of the deleted regions of 20q− is variable from patient to patient, although the critical deleted region had been determined in a 2·7 Mb region for myeloproliferative disease and a 2·6 Mb region for MDS/AML and a combined ‘myeloid’ common deleted region of 1·7 Mb (Bench et al, 2000). Recent studies suggested that loss or inactivation of one or more genes perhaps perturbs the regulation of haemopoietic progenitors, thus resulting in the genesis of clonal myeloid diseases (Asimakopoulos & Green, 1996). A set of genes were considered to be both positional and expression candidates for the target genes on 20q (Bench et al, 2000; MacGrogan et al, 2001).

The seven patients in this series had the following common clinical features. (i) All of them were older patients with a median age of 54 years. Meanwhile, we have analysed 43 de novo MDS patients with 20q− in our hospital. They were aged 49 ± 15 years old with a median age of 51 years. No statistical difference was found between these MDS patients with 20q− and those patients with i(20q−). (ii) They all had myeloid diseases: six with MDS, one with AML, which were diagnosed on haematological findings such as cytopaenia in peripheral blood and a hypercellular bone marrow with dysplastic changes involving bi- or tri-lineages. A mildly elevated HbF (18·9%) and ALIP were found in patient 3. These findings were also the evidence for the diagnosis of MDS. The features as mentioned in (i) and (ii) the same as those seen in MDS patients with 20q−. (iii) Their condition deteriorated rapidly with a short survival (16 months, 7 months, 12 d and 28 d for patients 1, 2, 4 and 5 respectively), and a high mortality (57%) except that three are still alive and anaemic. These facts indicate that MDS or AML patients with i(20q−) may possibly have a poor prognosis in contrast to MDS or AML patients with 20q−. As pointed out by Labal de Vinuesa et al (1987), the presence of an isochromosome in neoplastic cells could be related to exacerbation of the disease, which is probably caused by the selective growth advantage of cells with this abnormal chromosome. The true prognostic significance of this finding will require confirmation by the further study of such patients.

The molecular consequences of i(20q−) have not been clarified. There exist two possibilities in addition to loss or inactivation of one or more genes on 20q: the first possibility is the amplification of some oncogenes located on deleted 20q, due to the so-called gene dosage effect, and the second possibility is the deletion of the suppressor genes located on 20p. Further molecular study is necessary to elucidate the pathogenesis of myeloid diseases with i(20q−).

Acknowledgments

This work was supported by a grant (ZS0201) from the scientific and technological bureau of Suzhou.

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