Detection of mutator phenotype in Brazilian patients with acute and chronic myeloid leukemia

HUMAN AND MEDICAL GENETICS

RESEARCH ARTICLE

Detection of mutator phenotype in Brazilian patients with acute and chronic myeloid leukemia

Flávio Monteiro Ayres^{I, IV}; Euza Guimarães Momotuk^II; Celso da Cunha Bastos^III; Aparecido Divino da Cruz^IV

^IUniversidade Federal de Goiás, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia (Genética), Goiânia, GO, Brazil

^IIUniversidade Federal de Goiás, Instituto de Ciências Biológicas, Departamento de Biologia Geral, Goiânia, GO, Brazil

^IIIUniversidade Federal de Goiás, Faculdade de Medicina, Departamento de Clínica Médica, Goiânia, GO, Brazil

^IVUniversidade Católica de Goiás, Departamento de Biologia, Núcleo de Pesquisas Replicon, Goiânia, GO, Brazil

^{Correspondence}

ABSTRACT

The multisteps of tumorigenesis involve the classic chromosomal instability and the mutator phenotype pathways featured by a predisposition to acquire mutations in tumor suppressor genes and oncogenes. Expansion and contraction of microsatellite sequences due to a deficient mismatch repair system are a marker of the mutator phenotype. Controversial results regarding the extent of microsatellite instability (MSI) have been reported in the development and progression of myeloid malignancies. Here, we investigated MSI and loss of heterozygosity (LOH) frequencies at the microsatellite loci BAT-26, D7S486, D8S135, ANK1, IFNA, TP53 and bcr of 19 Brazilian patients with acute (AML) and chronic myeloid leukemia (CML). One AML patient and one CML patient were categorized as having a high degree of microsatellite instability (MSI-H), corresponding to 10.5% (2/19) of all patients. LOH at loci BAT-26 and TP53 was present in 30% of the patients with AML alone. Despite the small sample size, our results suggest that the mutator phenotype, as verified by MSI frequency, could play a role in the leukemogenesis of a small subset of patients with myeloid leukemia.

Key words: loss of heterozygosity, microsatellite instability, mismatch repair, mutator phenotype, myeloid leukemia.

Introduction

Leukemia is a proliferative disorder of the leukopoietic cells. The complex origin and nature of this disorder allow its classification into acute and chronic myeloid or lymphoid types (Harris et al., 2000). The multistep pathways of tumorigenesis involve the classic chromosomal instability pathway featured by chromosomal loss and LOH of tumor suppressor genes. In another possible pathway, a multistep mutator phenotype leads to the accumulation of mutations in tumor suppressor genes and oncogenes, due to a deficiency in the DNA mismatch repair system [MMR] (Peltomaki, 2001). Patients with such a predisposition to acquire mutations are likely to have cancer early in life, more than one primary tumor, and a family history of cancer (Ben-Yehuda et al., 1996).

Cells harboring a proficient MMR system can correct nucleotide mismatches during DNA replication or recombination and trigger apoptosis following serious DNA damage (Aquilina and Bignami, 2001). Deficiency to repair nucleotide mismatches causing loops in the template DNA strand during replication is associated to deletions of nucleotides in microsatellite loci. On the other hand, deficiency to repair the new DNA strand is related to the length expansion of microsatellite loci (Naidoo and Chetty, 1999). Microsatellites are highly polymorphic markers composed by short tracts of nucleotide repeats, dispersed throughout the human genome and prone to mutations by replication slippage. Increased frequency of somatic variation in the length of these markers, known as microsatellite instability (MSI), is a clear evidence of mutator phenotype (Aquilina and Bignami, 2001).

Controversial results regarding the extent of MSI have been reported in the development and progression of myeloid malignancies. This disagreement may be due to the number of patients studied, panel of markers chosen, MSI assessment criteria, source of non-tumoral control sample or methodological differences (Wada et al., 1994; Pabst et al., 1996; Mori et al., 1997; Tasaka et al., 1997; Boyer et al., 1998; Auner et al., 1999; Rimsza et al., 2000; Das-Gupta et al., 2001; Ribeiro et al., 2002). In this context, considering the scarcity of reports regarding such frequencies in Brazil, we aimed to investigate microsatellite alterations and the role of the mutator phenotype in a group of Brazilian patients with myeloid leukemia.

Materials and Methods

Patients and samples

Peripheral blood samples from 10 patients with acute and nine patients with chronic myeloid leukemia were collected in heparin at the Hematology Service of Hospital das Clínicas (Federal University of Goiás, Brazil). Additionally, exfoliated cells from the oral mucosa of every patient were obtained by gently scraping the inside lining of the mouth with a spatula. Patients were not selected based on stage, type nor therapy of the disease. Samples were collected from the patients following informed consent, according to the guidelines approved by the National Health Council of the Brazilian Ministry of Health.

DNA isolation

DNA samples were obtained from peripheral blood cells of all 19 patients and from bone marrow cells of two patients. DNA was isolated using conventional phenol-chloroform extraction and ethanol precipitation methods (Sambrook et al., 1989). Control DNA was extracted from the exfoliated buccal cells, using RapidPrep Micro Genomic DNA isolation Kit for Cells and Tissues (Amersham Biotech Inc., Piscataway, NJ) according to the manufacturer's instructions.

Microsatellite analysis

DNA amplification by polymerase chain reaction (PCR) was performed in a final volume of 25 µL containing 100 ng of genomic DNA, 50 mmol/L KCl, 10 mmol/L Tris HCl (pH 8.3), 1.5 mmol/L MgCl, 0.2 mmol/L each dNTP, 0.4 µmol/L of each sense and antisense primers for individual locus, and 1 U of Taq Polymerase (Amersham Biotech Inc.). A panel of seven microsatellite markers was used in this study (see Table 1). The touchdown thermal protocol included denaturation at 92ºC for 1 min, annealing at 58-48ºC (decreasing 1ºC/cycle for 10 cycles, followed by 15 cycles at 48ºC) for 1 min, and extension at 72ºC for 1 min. PCR products were visualized using an 8% polyacrylamide gel stained with 0.5 µ g/mL ethidium bromide. Gels were analyzed using a VDS system (Amersham Biosciences Inc., Umeå, Sweden) and the TotalLab software version 1.0 (Nonlinear Dynamics Ltd., Newcastle Upon Tyne, UK) to determine band sizes (bp) and densities. Decrease in the density of any given heterozygous allele was measured as arbitrary units of pixel concentration. The fractional allelic loss (FAL) was calculated using the formula FAL = CL/CI, where CL is the number of chromosome arms lost and CI is the overall number of informative chromosome arms.

Statistical analysis

Means of MSI or LOH were analyzed using Fisher's exact test with an overall significance level of 0.05 (95% CI) for comparisons between the two means.

Results

MSI and LOH were investigated for seven microsatellite markers scattered over six different chromosomes of AML and CML patients. MSI was identified as a variation in the length of microsatellite alleles in the leukemic sample, as compared to the corresponding constitutional material from buccal cells. Seven out of 109 paired PCR amplifications exhibited instability at the loci BAT-26, D7S486, D8S135, ANK1 and TP53 (Figure 1). Instabilities were verified in two patients with AML and two with CML, corresponding to approximately 21% (4/19) of patients with MSI . Two of these four patients had a high degree of MSI (MSI-H > 30% of assessed loci), suggesting the occurrence of the mutator phenotype. The other two patients with MSI had a low degree of MSI (MSI-L < 30% of assessed loci), suggesting the occurrence of another pathway of leukemogenesis rather than the mutator phenotype. Table 2 shows details of the results for each patient.

The criterion for LOH identification was the absence or a decrease to less than 50% in the intensity of one heterozygous band. The authors were aware of LOH misclassification due to technical artifacts, such as preferencial amplification or amplification failure due to DNA degradation and low amount of template. However, DNA preparations of good quality were available, and only heterozygous markers were considered informative. By using this criterion, markers bcr and D7S486 were heterozygous in most patients and, consequently, highly informative for LOH. Amplification of 31 paired sets of informative alleles from AML patients permitted the detection of two LOH events for marker TP53 and another two for marker BAT-26 (Figure 1). LOH events were found in three patients with AML, corresponding to 30% (3/10) of the AML patients. However, no LOH was detected in the 29 paired sets of informative alleles from CML patients (Table 2). Background data and statistic details for MSI and LOH are presented in Table 3.

Discussion

We studied 19 patients with myeloid leukemia and found 10.5% (2/19) of them with MSI–L and 10.5% (2/19) with MSI-H, which suggested the occurrence of the mutator phenotype in two of our patients (A11 and C07). It is noteworthy that MSI and LOH were not associated in any patient, corroborating the hypothesis that these two pathways of tumorigenesis do not occur together (Ponz de Leon et al., 1999). Additionally, it has been suggested that MSI is an adverse prognostic factor for leukemia and lymphoma patients, associated with short-term relapse and resistance to chemotherapy (Indraccolo et al., 1999). The association of mutator phenotype and TP53 molecular inactivation, as reported by Ben-Yehuda et al. (1996) and Zhu et al. (1999), might be an alternative pathway for de novo leukemogenesis in elderly people or for an accentuated level of genomic instability in therapy-related leukemia patients. Both hypotheses are thought to indicate a rather negative prognosis. Moreover, an increased incidence of adverse cytogenetic abnormalities has been found in AML patients with MSI, and even a patient with favorable cytogenetic findings, but with MSI, was reported to have had early relapse of AML (Das-Gupta et al., 2001). In contrast, in CML patients, MSI has been identified as an uncommon event (Mori et al., 1997; Auner et al., 1999; Ribeiro et al., 2002), although there is one former report suggesting that MSI may occur as a late event in the evolution of CML toward blast crisis (Wada et al., 1994).

In conformity with these previous findings, which correlate MSI and mutator phenotype with a more aggressive progression of AML, the prognosis of our AML patient (A11) with MSI-H was poor. The patient was initially diagnosed with myelodysplastic syndrome, which progressed to AML. Remission was successfully induced by chemotherapy, but the patient relapsed 17 months later and died during the second remission induction. On the other hand, our findings of MSI-H in CML were associated with a good prognosis. The CML patient C07 was treated with bulsulfan for six years, when the disease converted to the accelerated phase. The busulfan treatment was then replaced by hydroxyurea, and the disease has been under control for the last eight years. Cytogenetically this patient was found to be positive for the Ph chromosome, and bone marrow transplantation could not be done due to the lack of a histocompatible donor.

In the present study, the mean age of AML patients was 44 years (ranging from 18 to 68 years), and of CML patients 49 years (range: 34 to 76 years). Those of our patients who had MSI-H were 34 and 39 years old, while those who had MSI-L were 18 and 41 years old, respectively. It is of interest that our AML patients with MSI were younger than those studied by Das-Gupta et al. (2001), who reported that MSI in AML was restricted to elderly patients. It was suggested that exposure to environmental mutagens over a long period of time could lead to an increased frequency of MSI in elderly AML patients, due to defective MMR (Zhu et al., 1999; Das-Gupta et al., 2001). However, our findings of MSI-H were not associated with any history of chronical exposure to environmental mutagens.

All three new alleles found in the AML patients had greater lengths than those of the respective control sample, while CML patients had two larger and two smaller new alleles in the tumoral tissue, as compared to their corresponding wild-type controls (Figure 1). These new alleles with an increased length are an evidence of enhanced MMR deficiency in the new strand, immediately post DNA replication of leukemia cells (Naidoo and Chetty, 1999). This expansion of repetitive sequences may be part of a potential third tumorigenesis pathway characterized by epigenetic regulation. The epigenetic mechanisms regarding hematopoietic malignancies have been described as being likely to inactivate tumor suppressor genes by methylation of their promoter regions (Chim et al., 2002).

The absence of amplification of the BAT-26 locus of patient A28 suggested that a double allele deletion had occurred. Also, polymorphisms or mutations in the primer-binding site of one or both alleles could result in allele amplification failure or even the finding of null alleles. The possibility of amplification failure due to sample artifact was excluded after experimental repetitions and successful amplification of other six loci using the same DNA sample. Although the BAT-26 locus is an intronic site of the MMR gene hMSH2 (Rimsza et al., 2000), the lack of MSI in that patient may be due to some enzymatic overlap to repair DNA mismatches (Aquilina and Bignami, 2001). LOH of TP53 was found in our study with FAL values of 0.125 and 0.167 for patients A23 and A24, respectively. Once leukemia patients are highly expected to have chromosomal aberrations (Willman, 1999), the frequency of LOH in our study could be an underestimate, because only seven loci were surveyed to screen six different chromosomes. Additionally, contaminating normal cells could have obscured LOH assessment (Mori et al., 1997).

The MSI frequencies found in the present study were statistically different (P < 0.05, Fisher's exact test) from those found by Mori et al. (1997) , Boyer et al. (1998) , Auner et al. (1999) and Rimsza et al. (2000), who reported absence of MSI in myeloid leukemias. However, there was no statistical difference from some frequencies reported earlier by Wada et al. (1994), Pabst et al. (1996), Tasaka et al. (1997), Das-Gupta et al. (2001) and Ribeiro et al. (2002), whose findings demonstrated MSI in patients with myeloid leukemia. A statistical difference was also observed between the LOH frequency reported by Boyer et al. (1998) for the marker BAT-26 in AML patients and the frequency found in this study. However, there was no statistical difference between our LOH frequencies and those reported for loci D7S486 and TP53 (Mori et al., 1997) and also for some regions of the short arm of chromosome 2 described by Pabst et al. (1996).

In this and in most of the studies focusing on hematological malignancies, MSI was not a general phenomenon, supporting the idea that MSI is not suitable for differential diagnosis among different types of leukemia. Moreover, because tumor samples at diagnosis were not available, the roles of MSI and the mutator phenotype in the onset of myeloid leukemia in our patients were not clarified. Nonetheless, our findings regarding the mutator phenotype in myeloid leukemia suggest that this phenotype could play a role, if not in the onset and development, at least in the progression stage of leukemogenesis in a small subset of myeloid leukemia patients.

Acknowledgments

The authors thank Ana Márcia Fontes for collecting bone marrow samples and Fabiano R. Borges for helping with computer resources.

Received: February 15, 2003; Accepted: May 16, 2004

Associate Editor: Angela Maria Vianna-Morgante

• Aquilina G and Bignami M (2001) Mismatch repair in correction of replication errors and processing of DNA damage. J Cell Physiol 187:145-154.
• Auner HW, Olipitz W, Hoefler G, Bodner C, Konrad D, Crevenna R, Linkesch W and Sill H (1999) Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias. Br J Haematol 106:706-708.
• Ben-Yehuda D, Krichevsky S, Caspi O, Rund D, Polliack A, Abeliovich D, Zelig O, Yahalom V, Paltiel O, Or R, Peretz T, Ben-Neriah S, Yehuda O and Rachmilewitz EA (1996) Microsatellite instability and p53 mutations in therapy-related leukemia suggest mutator phenotype. Blood 1:4296-4303.
• Boyer JC, Risinger JI and Farber RA (1998) Stability of microsatellites in myeloid neoplasias. Cancer Genet Cytogenet 1:54-61.
• Chim CS, Liang R and Kowng YL (2002) Hypermethylation of gene promoters in hematological neoplasia. Hematol Oncol 20:167-176.
• Chissoe SL, Bodenteich A, Wang Y-F, Wang Y-P, Burian D, Clifton SW, Crabtree J, Freeman A, Iyer K, Jian L, Ma Y, McLaury H-JEN, Pan H-Q, Sarhan OH, Toth S, Wang Z, Zhang G, Heisterkamp N, Groffen J and Roe BA (1995) Sequence and analysis of the human ABL gene, the BCR gene, and regions involved in the Philadelphia chromosomal translocation. Genomics 27:67-82.
• Das-Gupta EP, Seedhouse CH and Russell NH (2001) Microsatellite instability occurs in defined subsets of patients with acute myeloblastic leukaemia. Br J Haematol 114:307-312.
• Goossens V, Sermon K, Lissens W, De Rycke M, Saerens B, De Vos A, Henderix P, Van de Velde H, Platteau P, Van Steirteghem A, Devroey P and Liebaers I (2003) Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the DeltaF508 mutation. Mol Hum Reprod. 9:559-567.
• Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, Lister TA and Bloomfield CD (2000) The world health organization classification of neoplasms of the hematopoietic and lymphoid tissues: report of the clinical advisory committee meeting Airlie House, Virginia, November, 1997. Hematol J 1:53-66.
• Indraccolo S, Minuzzo S, Nicoletti L, Cretella E, Simon M, Papakonstantinou G, Hehlmann R, Mion M, Bertorelle R, Roganovic J and Chieco-Bianchi L (1999) Mutator phenotype in human hematopoietic neoplasms and its association with deletions disabling DNA repair genes and bcl-2 rearrangements. Blood 1:2424-2432.
• Jones MH and Nakamura Y (1992) Detection of loss of heterozygosity at the human TP53 locus using a dinucleotide repeat polymorphism. Genes Chromosomes Cancer 5:89-90.
• Kwiatkowski DJ and Diaz MO (1992) Dinucleotide repeat polymorphism at the IFNA locus (9p22). Hum Mol Genet 1:658.
• Mori N, Morosetti R, Lee S, Spira S, Ben-Yehuda D, Schiller G, Landolfi R, Mizoguchi H and Koeffler HP (1997) Allelotype analysis in the evolution of chronic myelocytic leukemia. Blood 1, 2010-2014.
• Naidoo R and Chetty R (1999) DNA repair gene status in oesophageal cancer. Mol Pathol 52:125-130.
• Özcan R, Jarolim P, Lux SE, Ungewickell E and Eber SW (2003) Simultaneous (AC)_n microsatellite polyorphism analysis and single-stranded conformation polymorphism screening in an efficient strategy for detecting ankyrin-1 mutations in dominant hereditary spherocytosis. Br J Haematol 122:669-677.
• Pabst T, Schwaller J, Bellomo MJ, Oestreicher M, Muhlematter D, Tichelli A, Tobler A and Fey MF (1996) Frequent clonal loss of heterozygosity but scarcity of microsatellite instability at chromosomal breakpoint cluster regions in adult leukemias. Blood 1:1026-1034.
• Parsons R, Myeroff LL, Liu B, Willson JK, Markowitz SD, Kinzler KW and Vogelstein B (1995) Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer. Cancer Res 55:5548-5550.
• Peltomaki P (2001) Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum Mol Genet 10:735-740.
• Ponz de Leon M, Benatti P, Percesepe A, Rossi G., Viel A, Santarosa M, Pedroni M and Roncucci L (1999) Clinical and molecular diagnosis of hereditary non-polyposis colorectal cancer: problems and pitfalls in an extended pedigree. Ital J Gastroenterol Hepatol 31:476-480.
• Ribeiro EMSF, Rodriguez JM, Cóser VM, Sotero MG, Fonseca Neto JM, Pasquini R and Cavalli IJ (2002) Microsatellite instability and cytogenetic survey in myeloid leukemias. Braz J Med Bio Res 35:153-159.
• Rimsza LM, Kopecky KJ, Ruschulte J, Chen IM, Slovak ML, Karanes C, Karanes C, Godwin J, List A and Willman CL (2000) Microsatellite instability is not a defining genetic feature of acute myeloid leukemogenesis in adults: Results of a retrospective study of 132 patients and review of the literature. Leukemia 14:1044-1051.
• Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory Press. New York, E.3pp.
• Tasaka T, Lee S, Spira S, Takeuchi S, Nagai M, Takahara J, and Koeffler HP (1997) Microsatellite instability during the progression of acute myelocytic leukaemia. Br J Haematol 98:219-221.
• Wada C, Shionoya S, Fujino Y, Tokuhiro H, Akahoshi T, Uchida T, and Ohtani H (1994) Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia. Blood 15:3449-3456.
• Willman CL (1999) Molecular evaluation of acute myeloid leukemias. Semin Hematol 36:390-400.
• Wood S, Mitchell HK and Schertzer M (1991) Isolation and analysis of dinucleotide repeat polymorphisms from a flow-sorted chromosome 8 library. Cytogenet Cell Genet 58:1932.
• Zhu YM, Das-Gupta EP and Russell NH (1999) Microsatellite instability and p53 mutations are associated with abnormal expression of the MSH2 gene in adult acute leukemia. Blood 15:733-740.

Publication Dates

• Publication in this collection
  14 Jan 2005
• Date of issue
  2004

History

• Received
  15 Feb 2003
• Accepted
  16 May 2004