Chinese Medical Journal 2003;116(9):1298-1303
Chromosomal changes detected by fluorescence in situ hybridization in patients with acute lymphoblastic leukemia

ZHANG Lijun 张丽君,  PARKHURST JB,  KERN WF,  SCOTT KV,  NICCUM D,  MULVIHILL JJ,  LI Shibo 李师伯

ZHANG Lijun 张丽君 (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA;Department of Hematology, the First Affiliated Hospital of China Medical University, Shenyang 110001, China)

PARKHURST JB (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

KERN WF (Department of Pathology, University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

SCOTT KV (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

NICCUM D (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

MULVIHILL JJ (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

LI Shibo 李师伯 (Departments of Pediatrics,University of Oklahoma, Health Sciences Center, the Oklahoma, OK 73104, USA)

Correspondence to:Shibo Li,Department of Pediatrics, Health Sciences Center, University of Oklahoma, OK 73104, USA (Tel: 001-405-2713590. Fax:. E-mail:)
Keywords
acute lymphoblastic leukemia;fluorescence in situ hybridization;chromosome rearrangements
Abstract
Objectives To investigate patients with acute lymphoblastic leukemia (ALL) for TEL/AML1 fusion, BCR/ABL fusion, MLL gene rearrangements, and numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ hybridization (FISH) and to determine the relationship and the significance of those findings.
Methods Fifty-one American patients (34 men and 17 women) were included in this study. Of them there were 41 patients with pro-B cell type ALL, 9 with B cell type ALL and 1 with T cell type ALL. Chromosome metaphases of each sample were prepared according to standard protocols. Fluorescence in situ hybridization was performed using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, specific chromosome centromere probes and dual color/multiple color translocation fusion probes. The digital image analysis was carried out using Cytovision and Quips FISH programs.
Results An overall incidence of chromosomal anomalies, including t (9;22), MLL gene rearrangements, t (12;21), and numerical chromosomal anomalies of chromosomes 4, 10, 17 and 21 was found in 33 patients (65%). Thirty-one of them were pediatric patients and two adults. The t (12;21) was the commonest chromosomal anomaly detected in this population; 14 out of the 45 pediatric patients (31%) were positive for TEL/AML1 fusion, among which three had an additional derivative 21 [t (12;21)], four had a deletion of 12p and two had an extra copy of chromosome 21. All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping. The percentage of cells with fusion signals ranged from 20% to 80%. All fourteen patients positive for TEL/AML1 gene fusion were mosaic. Three out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. All pediatric patients with pre-T or T cell lineage and the six adults were negative for TEL/AML1 fusion. One patient had double Philadelphia chromosomes, three had a rearrangement or a deletion of the MLL gene, one had t (4;11) and two had a deletion of the MLL. One of the patients with an MLL deletion also had a large ring of chromosome 21, and r (21) was caused by AML1 gene tandemly duplicated at least five times. The second case with the MLL deletion was also unique, the patient had a t (12;21) as well. A total of 20 patients had numerical changes (gain or loss) of chromosomes 4, 10, 17 and 21. Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ABL or the MLL gene rearrangement; one did have the TEL/AML1 gene fusion. Eleven patients with pro-B cell or B cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21), among them, 10 patients had no structural alteration of chromosome 21, and one was combined by t (12; 21). Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1.
Conclusions FISH plays an important role in detecting chromosome changes, especially in some cryptic chromosome translocations and patients with culture failures. This study found a trend towards a division between patients who had structural changes such as t (12;21) or a ring chromosome 21 and those who had numerical changes of chromosome 21 as well as the patients with TEL/AML1 fusion and patients with the coexistence of numerical chromosomal changes of chromosomes 4, 10 and 17. In our opinion there are two separate mechanisms which lead to the development or progression of leukemia.

Most of our current understanding of the biological and clinical significance of genetic alterations in ALL rely on the new techniques developed recently. Conventional cytogenetics (CC) enabled the description of most of the recurrent chromosomal changes to occur in ALL.[1-4] It is one of the standard tests for confirming of the clinical diagnosis, classification, treatment and prognosis of patients with ALL. However, CC has many limitation, especially in ALL. In fact, cytogenetic techniques are often hampered by a low yield or poor quality of the metaphases available from bone marrow or leukemic blood. It can be easy to miss chromosomal abnormalities. On the other hand, some cryptic chromosomal translocations are impossible to detect by conventional cytogenetic methods. One example is the TEL/AML1 fusion resulting from a cryptic translocation between chromosomes number 12 and 21 [t (12;21) (p13; q22)]. This translocation is impossible to be identified by conventional cytogenetic analysis, but can be readily detected using fluorescence in situ hybridization analysis (FISH) techniques with commercially available DNA probes. It became evident that some chromosomal changes had a major prognostic value: hyperdiploidy and t (12;21) are associated with very long disease free survival, and the gain of chromosomes 4, 10, 17 and 21 is related to a good prognosis, whereas t (9;22) and 11q23 rearrangements correlate with a poor outcome.[5,6] Because of this prognostic impact, detection of these specific abnormalities has to be accurate and rapid at diagnosis. In this study, we have used a combination of multiple DNA probes to evaluate fifty-one patients with ALL to determine the frequency of chromosomal anomalies and the relationship as well as the significance of those findings.                          

METHODS

Patients
A total of fifty-one American patients (34 men and 17 women) were included in the study; forty-five of them were pediatric patients (6 months to 19 years old) and the remaining were adults (23 to 74 years old). They were initially referred for routine chromosome studies during 1995-2002. Every available specimen with a clinical diagnosis of ALL was included in the study without an ascertainment bias. Fluorescence in situ hybridization analysis
FISH analysis was performed on interphase and/or metaphase cells using multiple commercially available probes (Vysis Inc., Downers Grove, IL, USA). These include the LSI TEL/AML1 ES dual color translocation for t (12;21), the probe for MLL at 11q23 and the LSI BCR/ABL ES dual color translocation probe for t (9;22). The LSI TEL/AML1 ES dual color translocation probe is a mixture of the LSI TEL probe labeled with SpectrumGreen and the LSI AML1 probe labeled with SpectrumOrange. LSI MLL is a break apart rearrangement dual color DNA probe, located at 11q23; the centromeric side of the MLL gene is labeled with SpectrumGreen and the telomeric side of the MLL gene is labeled with SpectrumOrange. LSI BCR/ABL ES dual color fusion translocation probe is a mixture of the LSI BCR probe labeled with SpectrumGreen and the LSI ABL probe labeled with SpectrumOrange. The alpha satellite DNA probes for numerical changes include chromosomes 4, 10, and 17. The AML1 gene also served as a DNA marker to detect numerical chromosomal changes of chromosome 21. All probes were designed to detect specific chromosomal changes in both metaphase and interphase cells. Images were acquired using an Axioskop fluorescence microscope equipped with CytoVison Software from Applied Imaging, Inc., USA. A total of 60 to 100 cells were analyzed from each specimen. At least 5% of cells with abnormal hybridization pattern/signals were considered as the minimum cut-off level. RESULTSThirty-three out of 51 patients (65%) had abnormal FISH findings ( Table ). One of them had a positive screen for BCR/ABL fusion. This patient was found to have four fusion signals, i.e. two derivative chromosomes 9 and two derivative chromosomes 22; a very rare phenomenon called double Philadelphia chromosomes ( Fig. 1 ). Routine chromosome analysis showed that the patient had complex chromosomal changes; in addition to Philadelphia chromosomes, the majority of cells analyzed were hyperdiploid (chromosome numbers ranging from 46 to 65), had a tandem duplication of a chromosomal segment of chromosome 1 (q21 to q32), and additional material of unknown chromosomal origin attached to band 11q23 (data not shown). A total of three patients had MLL gene rearrangements. Of them, one had a split signal of the MLL gene caused by a translocation between chromosomes 4 and 11, t (4;11), found by conventional cytogenetic analysis (data not shown), two had a deletion of the MLL gene. One of the patients with an MLL deletion also had a large ring of chromosome 21, this was ascertained when searching for the TEL/AML1 translocation utilizing the TEL/AML1 FISH probe. At least five to six signals of the AML1 gene were detected on the ring chromosome 21, indicating that the chromosome 21 material covered by the AML1 gene region was tandemly duplicated at least five times ( Figs. 2 and 3 ). As far as we know, this is the first case with multiple duplications of the AML1 gene in the form of a ring chromosome. Another patient with the MLL deletion was also unique, the patient had a t (12; 21) as well ( Table ). Fourteen out of 45 pediatric patients (31%) were positive for TEL/AML1 fusion; all six adults were negative. All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping; thirteen were newly diagnosed with leukemia, and one was in a second relapse, 9 years after the first episode of ALL. The percentage of cells with fusion signals ranged from 20 to 80 percent. All fourteen patients positive for TEL/AML1 gene fusion were mosaic; i.e. some cells had a normal TEL/AML1 hybridization pattern, and some cells had a fusion signal of TEL/AML1 ( Fig. 4 ). Three of those fourteen patients had two fusion signals ( Fig. 5 ) and four had only one signal for the TEL gene ( Fig. 6 ), this implied that one TEL gene was deleted. It is worthwhile to mention that 3 out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. Eleven patients with pro-B cell or B cell type ALL ( 9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21), among them, 10 patients had no structural alteration of chromosome 21, and one patient was combined by t (12; 21). A total of 20 patients with B or pre-B cell leukemia had numerical changes (gain or loss) of chromosomes 4, 10, 17 or 21 ( Table ). Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ABL or the MLL gene rearrangement; one did have the TEL/AML1 gene fusion. Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1.

DISCUSSION

Nonrandom chromosomal changes were found in more than 80% of patients with ALL and were used as independent prognostic indicators.[7,8] The changes include numerical chromosomal anomalies and structural rearrangements. In most cases, patients have both at the same time. A better prognosis is expected in patients with t (12; 21) (p13; q22) and hyperploidy (50-65 chromosomes). Patients with the TEL/AML1 fusion or gain of chromosomes 4, 10, 17 and 21 may have a particularly low risk of treatment failure.[9-11] It is reported that there is a relationship between chromosome trisomies and chemotherapy in patients with ALL, and chromosome 4, 10, 17, 21 trisomies is related to a good prognosis,[12-16] while poor prognosis is commonly seen in patients with t (9; 22) (q34; q11.2), MLL gene rearrangements and hypoploidy (23-29 chromosomes).[17,18] Risk-directed therapy to patients according to biological factors is the current therapeutic strategy to maximize clinical efficacy and to minimize treatment-related toxicity. Identifying these changes will provide accurate diagnostic and prognostic information and better patient treatment and management. We tried to detect chromosome changes related to prognosis of patients with ALL by using a combination of multiple FISH probes. In our series of 51 patients, fourteen patients with the TEL/AML1 fusion and eight patients with a gain of at least three of the four chromosomes, 4, 10, 17 or 21, one patient had both the TEL/AML1 fusion and a gain of chromosomes 4, 10 and 21. Eleven patients with pro-B cell or B cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21), among them, 10 patients had no structural alteration of chromosome 21, and one patient was combined by t (12; 21). A total of 41% of the patients were positive for the TEL/AML1 fusion or a gain of chromosomes. This study found a trend toward a division between patients who had structural changes such as t (12; 21) or a ring chromosome 21 and those who had numerical changes of chromosome 21 as well as the patients with TEL/AML1 fusion and patients with the coexistence of numerical chromosomal changes of chromosomes 4, 10 and 17. We considered that there are two separate mechanisms which lead to the development or progression of leukemia. In order to draw a conclusion concerning the correlation between clinical and laboratory findings, all patients in our study are closely followed up.

Four patients were found to have chromosomal changes associated with poor prognosis; the first patient had double BCR/ABL fusions and died 4 years after diagnosed as having ALL, the second patient had a split signal of the MLL gene caused by a translocation between chromosomes 4 and 11, and the third patient had a deletion of the MLL gene and the large ring chromosome 21. They all experienced resistance to chemotherapy and had less than 5 years survival after the diagnosis of ALL. The last patient was an interesting one, he had both the deletion of the MLL gene and the TEL/AML1 gene fusion. The patient was diagnosed with ALL two years ago and is now in complete remission. It is unclear how long the remission will last. The patient needs to be clinically followed up to determine which biological character plays a main role in the ALL development when two opposite predictors appear in the same case. We also observed that four patients had a deletion of the TEL gene. Two of the four patients had B-cell lineage leukemia. The significance of these findings is unknown. It will be interesting to further investigate whether TEL gene deletion is a potential chromosome marker to predict the outcome of treatment in patients with B-cell ALL. Our data suggest that FISH testing using DNA probes specific for TEL/AML1, BCR/ABL, MLL, and alpha satellite DNA probes 4, 10, and 17 is a powerful tool for leukemia and it should be a routine procedure for all patients with newly diagnosed ALL. They will detect the majority of consistent chromosomal anomalies that may have a value in predicting treatment outcome, and aid in preventing highly curable patients from experiencing unnecessary side effects of the chemo/radiation therapies.


Acknowledgements: We would like to express our thanks to the laboratory technologists and Dr. Razia S. Muneer for the previous cytogenetic studies of some of the patients.

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