Introduction

MDS constitutes a group of hematological disorders characterized by peripheral blood cytopenias; secondary to bone marrow dysfunction, MDS occurs predominantly in adult patients (usually >60 years of age) and evolves in AML¹ in about 30% of the cases after variable intervals from diagnosis. The clinical transition is demonstrated by the clonal proliferation of the hematopoietic precursor that generates leukemic blasts unable to differentiate. It is considered that the evolution to AML is associated with additional genetic changes acquired by MDS patients. Moreover, AML evolving from MDS is much less responsive to chemotherapeutic agents than is de novo AML.²

Approximately half of MDS patients have a detectable chromosome abnormality, usually a total or partial deletion of chromosome 5 or 7 and/or trisomy 8, but translocations and amplifications are not very frequent. Allelic loss has been found on chromosomes 6q, 7p, 10p, 11q, 14q and 20q, and, even if there is no specific relationship between most of the rearrangements and the clinical outcome, MDS patients with abnormal karyotype are usually thought to be at a higher risk for developing AML than MDS patients having normal karyotype.^3,4 Nevertheless, the management of MDS patients showing normal karyotype by means of classic cytogenetic techniques is still a problem. It has recently been observed that the clinical follow-up of these patients is not sufficient since some of them have surprisingly worse and poorer clinical outcome than expected.^5,6 Therefore, it seems of great interest to detect prognostic elements to add to the karyotype in MDS, in order to identify higher risk patients.

The human gene of PI-PLC 1 has been recently characterized.⁷ PI-PLC 1 is a signaling molecule involved in the control of cell cycle, and is also present in the nucleus.⁸ PI-PLC1 gene, constituted from 36 small exons and introns, has been located⁹ on the short arm of human chromosome 20 (20p12, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene. The chromosome band 20p12 has been shown to be rearranged in human diseases such as solid tumours without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes.^10,11,12,13

Here we report evidence that deletion of PI-PLC1 gene is present in MDS patients rapidly evolved to AML.

Materials and methods

Patients

The study has been performed screening different groups of patients affected with MDS and/or AML. Bone marrow and cytogenetic specimens were from the Department of Hematology of the General Hospital of Pescara.

The first group (Group 1) has been constituted by patients with hematological disorders and karyotype abnormalities revealed at GTG banding. Within this group, we chose five patients showing at spectral karyotype (SKY) analysis rearrangements involving chromosome 20. Two patients, 80 and 77 years old, suffered, respectively, for MDS and refractory anemia with excess of blasts in transformation (patients 1 and 2). These two patients did not receive therapy but only supportive care, and died 9 and 6 months, respectively, after the diagnosis. The other three patients affected by AML had cytosine-arabinoside treatment without acquiring the remission and died between 5 and 7 months after the diagnosis.

The second group (Group 2) has been constituted by patients diagnosed with AML (six patients) or MDS (nine patients); all these patients had standard GTG-banding karyotype at diagnosis that resulted normal. MDS patients received supportive care, with no treatment excepting for those who developed AML. The AML-affected patients have been treated with standard protocol with chemotherapeutic drugs.

Cytogenetic, SKY and FISH analyses

Cytogenetic investigations were carried out on unstimulated bone marrow cells incubated for 24 h. High-resolution (>500 bands) GTG-banded metaphase chromosomes were prepared according to standard protocols.¹⁴

In the first group of patients (Group 1), SKY analysis has been performed in order to identify the chromosome imbalances not identified by standard cytogenetics. SKY experiments were performed according to Schrock et al¹⁵ and 10 metaphases were used for spectral analysis. FISH analysis was performed according to Calabrese et al¹⁶ by using specific probes for the gene of PI-PLC1 (PAC clone HS881E24 from P de Jong RPCI-5 PAC library) and a probe for chromosome 20p arm subtelomeric region (Cytocell/Celbio, Italy).

In the second group of patients (Group 2), we performed FISH analysis by using the same PI-PLC1 probe as above and subtelomeric 20p probe (Cytocell/Celbio, Italy), painting FISH analysis for chromosome 20 (Oncor/Resnova, Italy) and FISH analysis by using PI-PLC4 cDNA probe (kindly provided by Dr Sue Goo Rhee, NIH, Bethesda, MD, USA) and subtelomeric 20p probe (Cytocell/Celbio, Italy). FISH analysis was performed according to standard methods; the DNA probe has been biotin labeled by nick translation and detected with Cy3-conjugated streptavidin (Sigma-Aldrich). The painting FISH analysis was performed according to the manufacturer's data. The images were acquired by using a Nikon Eclipse 800 fluorescence microscope and the Genikon system.

Immunocytochemical analysis

The immunocytochemical analysis has been performed in the second group (Group 2) of normal karyotype patients by using an anti-PI-PLC 1 monoclonal antibody revealed with a secondary anti-mouse antibody Cy-3 conjugated (Santa Cruz Technologies, Santa Cruz, CA) according to the manufacturer's protocol, and the images were acquired by using a Nikon Eclipse 800 fluorescence microscope and the ACT-1 Nikon system.

Results and discussion

In the first group of patients (Group 1) affected with different hematological disorders and complex karyotype for the presence of complex chromosome rearrangements and indecipherable markers, we performed SKY analysis for a more accurate identification of the karyotype which disclosed rearrangements of chromosome 20 consisting in total or partial gains or losses in five patients. In those five patients, SKY analysis (Figure 1) disclosed 39 structural chromosome changes (Table 1). All the five patients had an MDS at onset. In all the five patients SKY disclosed chromosome aberrations consisting of simple or complex changes, a translocation involving the long arm of chromosome 13 (13q) and the chromosome 20 [t(13;20)] being observed in three of them, that is, patients 1, 4 and 5. Using a specific probe for the PI-PLC1 gene, recently mapped on 20p12,⁹ FISH analysis disclosed the loss of one allele of the gene in all those three patients, allowing the localization of the breakpoint on the short arm of chromosome 20 (Figure 1). In patients 2 and 3, SKY showed, respectively, a t(11;20) with break point at band 11q23 (patient 2) and a t(17;20) with break at 17q11 (patient 3). Also, in the latter two cases, FISH analysis showed the monoallelic deletion of the PI-PLC1 gene allowing the localization of the breakpoint at band 20p12 (Table 1). Moreover, in patient 2, FISH analysis by using specific probes for CALM (OMIM ^*603025 located on 11q14) and MLL (OMIM ^*159555; located on 11q23) genes, frequently rearranged in AML karyotype,^17,18 showed that the two genes were not rearranged because of the translocation. In patient 3, bearing the translocation between chromosomes 17 and 20, the deletion of PI-PLC1 affected both alleles. The immunocytochemical analysis on blast nuclei from a cytogenetic sample of this patient showed a complete lack of reactivity of the monoclonal antibody, which recognizes PI-PLC1 (not shown, in that there is an uniform dark field).

Figure 1.

SKY and FISH analysis (by using subtelomeric 20pter probe (green) and PI-PLC1 probe HS881E24 (red)) carried out on cytogenetic samples from patient no 5 (Group 1). For clinical and cytogenetic data, see Table 1. The FISH analysis is representative of all the patients bearing PI-PLC1 gene deletion.

Full figure and legend (157K)

Table 1 - Patients characteristics (Group 1).

Full table

Rearrangements of the short arm of chromosome 20 have been detected in a number of patients with solid tumors, but rarely in hematological disorders.^{10,11,12,13,19} In all the five patients, the 20p rearrangement was associated with the deletion of PI-PLC1 gene, whose product is implicated in signal transduction and cell differentiation.^20,21 Nevertheless, the association with other chromosome aberrations hampers the definition of the role played by the 20p abnormalities in both the onset and the evolution of the disease. Therefore, we focused our attention on the second group of patients (Group 2; Table 2) with normal high-resolution GTG-banding karyotype. The FISH analysis with the probe for PI-PLC1 (Figure 2) in six AML patients showed that two of them had a monoallelic deletion of the gene. These two patients died in a time frame ranging from 1 to 12 months after the diagnosis. The FISH analysis in nine patients affected with MDS, having normal high-resolution GTG-banding karyotype, showed that four of them had a monoallelic deletion of the gene (Figure 2). All of these patients died in a time frame ranging from 1 to 6 months after developing AML. The patient who died 4 months after the diagnosis had a secondary MDS after the 4 years that he had received therapy for Hodgkin disease; this is particularly interesting, as it is well known that secondary MDS are at high risk for developing leukemia. The total painting for chromosome 20 resulted normal in all the 15 analyzed patients.

Figure 2.

FISH analysis carried out on cytogenetic samples from patients with normal standard karyotype (Group 2): (a) AML patient: FISH by using subtelomeric 20pter probe (green) and PI-PLC1 probe HS881E24 (red); only one copy of chromosome 20 shows both signals, as the other shows deletion of the PI-PLC1 gene; (b) MDS patient: FISH by using subtelomeric 20pter probe (green) and PI-PLC1 probe HS881E24 (red); only one copy of chromosome 20 shows both signals, as the other shows deletion of the PI-PLC1 gene; (c) Normal control: FISH by using subtelomeric 20qter probe (green) and PI-PLC1 probe HS881E24 (red); both copies of the chromosome 20 show both signals. The FISH analysis is representative of all the patients bearing PI-PLC1 gene deletion.

Full figure and legend (112K)

Table 2 - Patients characteristics (group 2).

Full table

To establish the amplitude of the deletion and the possible involvement of genes other than PI-PLC1 within the 20p12 region, we used a probe for another gene localized in the same band, that is, the PI-PLC4 gene, located at less than 1 Mb from PI-PLC1 (http://www.ensembl.org/Homo_sapiens/mapview?chr=20). PI-PLC4 is another member of the PI-PLC family, which in mammals is specifically expressed in the nervous system and is involved in retinal phototransduction,²² while PI-PLC1 is mainly involved in the control of cell cycle.²³ Performing FISH analysis by using a cDNA probe for PI-PLC4 (Figure 3), we found that five of the six patients bearing the monoallelic deletion of PI-PLC1 were normal, while one of them, patient 13, had also the monoallelic deletion of PI-PLC4. This result indicates that in this patient the region comprised between the two genes with a deletion spanning from 1 to 5 Mb has been lost (the resolution of standard high-resolution karyotype). The results of the remaining five patients indicate that the absence of one allele of PI-PLC1 gene could be due to an interstitial deletion, which does not affect the PI-PLC4 gene located 0.1 Mb far from PI-PLC1.

Figure 3.

FISH analysis carried out on cytogenetic samples from patients with normal standard karyotype (Group 2): patient no. 9. FISH by using probes for PI-PLC1 (red) and subtelomeric 20pter (green) (a) shows the deletion of one allele, while, by using probes for PI-PLC 4 and subtelomeric 20pter (b), shows both signals; for clinical and cytogenetic data, see Table 2. The FISH analysis is representative of all the patients bearing PI-PLC1 gene deletion.

Full figure and legend (99K)

By performing, in group 2 of patients, the immunocytochemical analysis using an anti-PI-PLC1 antibody (Figure 4), we found that all the AML/MDS patients who resulted normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PI-PLC1.²⁴ All the AML/MDS patients bearing the monoallelic deletion of PI-PLC1 gene show reduced signal intensity when compared to normal control images using the same time of exposure (Figure 4). We should take into account that, in the AML patient number 3 of group 1 with chromosome 20 rearrangements, the loss of both alleles resulted in a complete lack of PI-PLC1 expression.

Figure 4.

Immunocytochemical analysis carried out on cytogenetic samples from patients with normal standard karyotype. Blast nuclei were stained with an antibody Cy3-conjugated; normal control (a), AML (b) and MDS (b) patients. (a) and (b) patients, bearing the deletion of the PI-PLC1 gene, show reduction in the intensity of the signal compared to normal control. The bone marrow blast percentages in AML patients were ranging from 30 to 80%. The bone marrow blast percentages in MDS patients were ranging from 10 to 18%. The immunocytochemical analysis is representative of all the patients bearing PI-PLC1 gene deletion.

Full figure and legend (58K)

The clinical evolution and the progression of the disease of the MDS patients, all showing at diagnosis a normal karyotype and considered for this reason at good prognosis, has been worser than expected, as all of them developed AML and died in a time frame ranging from 1 to 6 months. Moreover, the normal karyotype MDS patients, showing nondeletion of PI-PLC1 gene at FISH analysis, are still alive at least at 24 months after the diagnosis. All in all, the genetic anomaly affecting a key signaling PI-PLC, which is capable of controlling the Cyclin D3/cdk4 checkpoint in G1 phase,²³ hints at a possible role in the pathophysiology of MDS and gives a first clue for the likelihood that PI-PLC1 could be involved in the progression of the disease.

This is obviously a preliminary observation based on a small group of patients, and in our opinion it offers the rationale for the analysis of a large series of patients with uniform clinical approach in order to prove that deletion of PI-PLC 1 gene could provide a new tool to discriminate a high-risk group within normal karyotype MDS patients, who are usually classified as at low risk for developing leukemia.

References

1. Silverman LR. Myelodysplastic syndrome. In: Bast RC, Kufe DW, Pollock RE, Weichselbaum RR, Holland JF, Frei E (eds). Cancer Medicine, 5th edn. Hamilton (Canada): BC Decker Inc. Publ., 2000, pp. 123.
2. Zeidman A, Dayan DB, Mittelman M. Secondary myelodysplastic syndromes and acute leukemias. Haematologia 1995; 27: 23–28. | PubMed |
3. Mrózek K, Heinonen K, de la Chapelle A, Bloomfield CD. Clinical significance of cytogenetics in acute myeloid leukemia. Semin Oncol 1997; 24: 17–31. | PubMed | ISI | ChemPort |
4. Bloomfield CD, Shuma C, Regal L. Long-term survival of patients with acute myeloid leukemia. A third follow-up of the Fourth International Workshop on Chromosomes in Leukemia. Cancer 1997; 80 (Suppl): 2191–2198. | Article | PubMed | ISI | ChemPort |
5. Caligiuri MA, Bloomfield CD. The molecular biology of leukemia. In: DeVita Jr VT, Hellman S, Rosenberg SA (eds). Cancer: Principles & Practice of Oncology, 6th edn. Philadelphia, PA: Lippincott-Raven, 2000, pp. 2389–2404.
6. Sheikhha MH, Awan A, Tobal K, Liu Yin JA. Prognostic significance of FLT3 ITD and D835 mutations in AML patients. Hematol J 2003; 4: 41–46. | Article | PubMed |
7. Peruzzi D, Aluigi M, Manzoli L, Billi AM, Di Giorgio FP, Morleo M et al. Molecular characterization of the human PLC1 gene. Biochim Biophys Acta 2002; 1584: 46–54. | Article | PubMed |
8. Martelli AM, Manzoli L, Faenza I, Bortul R, Billi A, Cocco L. Nuclear inositol lipid signaling and its potential involvement in malignant transformation. Biochim Biophys Acta (Rev. Cancer) 2002; 1603: 10–16.
9. Peruzzi D, Calabrese G, Faenza I, Manzoli L, Matteucci A, Gianfrancesco F et al. Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C1. Biochim Biophys Acta 2000; 484: 175–182.
10. Hu J, Jiang CC, Ng HK, Pang JC, Tong CY, Chen SQ. An allelotype study of primary and corresponding recurrent glioblastoma multiforme. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2003; 20: 56–58. | PubMed |
11. Gordon A, McManus A, Anderson J, Fisher C, Abe S, Nojima T et al. Chromosomal imbalances in pleomorphic rhabdomyosarcomas and identification of the alveolar rhabdomyosarcoma-associated PAX3–FOXO1A fusion gene in one case. Cancer Genet Cytogenet 2003; 140: 73–77. | Article | PubMed |
12. Peng Z, Zhou C, Zhang F, Ling Y, Tang H, Bai S et al. Loss of heterozygosity of chromosome 20 in sporadic colorectal cancer. Chin Med J (Engl) 2002; 115: 1529–1532. | PubMed |
13. Wada N, Duh QY, Miura D, Brunaud L, Wong MG, Clark OH. Chromosomal aberrations by comparative genomic hybridization in hurthle cell thyroid carcinomas are associated with tumor recurrence. J Clin Endocrinol Metab 2002; 87: 4595–4601. | Article | PubMed |
14. Le Beau MM. Cytogenetics Analysis of Hematological Malignant Diseases. In: Barch MJ (ed). The ACT Cytogenetics Laboratory Manual, Chapter 9, 2nd edn. New York, NY: Raven Press, 1991, pp. 395–449.
15. Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA et al. Multicolor spectral karyotyping of human chromosomes. Science 1996; 273: 494–497. | Article | PubMed | ISI | ChemPort |
16. Calabrese G, Fantasia D, Spadano A, Morizio E, Di Bartolomeo P, Palka G. Karyotype refinement in five patients with acute myeloid leukemia using spectral karyotyping. Haematologica 2000; 85: 1219–1221. | PubMed |
17. Calabrese G, Sallese G, Stornaiuolo A, Morizio E, Palka G, De Blasi A. Assignment of the beta-arrestin 1 gene (ARRB1) to human chromosome 11q13. Genomics 1994; 23: 169–171. | Article |
18. Castro PD, Fairman J, Nagarajan L. The unexplored 5q13 locus: a role in hematopoietic malignancies. Leuk Lymphoma 1998; 30: 443–448. | PubMed |
19. Mori N, Morosetti R, Hoflehner E, Lubbert M, Mizoguchi H, Koeffler HP. Allelic loss in the progression of myelodysplastic syndrome. Cancer Res 2000; 60: 3039–3042. | PubMed |
20. Martelli AM, Gilmour RS, Bertagnolo V, Neri LM, Manzoli L, Cocco L. Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells. Nature 1992; 358: 242–245. | Article | PubMed | ISI | ChemPort |
21. Cocco L, Martelli AM, Barnabei O, Manzoli FA. Nuclear inositol lipid signaling. Adv Enzyme Regul 2001; 41: 361–384. | Article | PubMed |
22. Alvarez RA, Ghalayini AJ, Xu P, Hardcastle A, Bhattacharya S, Rao PN et al. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C beta 4 (PLCB4). Genomics 1995; 29: 53–61. | Article | PubMed | ISI | ChemPort |
23. Faenza I, Matteucci A, Manzoli L, Billi AM, Aluigi M, Peruzzi D et al. A role for nuclear phospholipase C1 in cell cycle control. J Biol Chem 2000; 275: 30520–30524. | Article | PubMed | ISI | ChemPort |
24. Faenza I, Bavelloni A, Fiume R, Lattanzi G, Maraldi NM, Gilmour RS et al. Up-regulation of nuclear PLCbeta1 in myogenic differentiation. J Cell Physiol 2003; 195: 446–452. | Article | PubMed |

Acknowledgements

Acknowledgments for research support: This work was supported by grants from: AIRC, CARISBO Foundation, Italian MIUR Cofin, Italian CNR-MIUR Oncology Project.