Introduction

One-third of leukemia patients have specific chromosome translocations that are closely associated with certain clinical and biological features.¹ Since translocations are the most significant prognostic factor in leukemia, cytogenetic analysis is essential for the initial checkup of patients with leukemia.¹ In addition, in certain subgroups of leukemia classified by the age of patients or the morphology of leukemic cells, molecular analysis using the Southern blot or reverse transcription-polymerase chain reaction (RT-PCR) method detected specific translocations more frequently than cytogenetic analysis.^2,3,4,5,6

Infant leukemia is associated with particular translocations, most commonly 11q23 translocation.^4,6 The MLL gene (also known as ALL-1 or HRX) that was isolated from the breakpoint region of 11q23 translocation is fused to more than 50 different partner genes.⁷ While cytogenetic analysis detects 11q23 translocation in 63 and 43% of infant acute lymphoblastic leukemia (ALL) and infant acute myeloid leukemia (AML), respectively, Southern blot analysis detects MLL rearrangements in 82 and 63% of respective leukemias.^4,6 Contradictions between cytogenetic and Southern blot findings on 11q23/MLL rearrangements were also reported in adult patients with AML-M4/M5.³ There have been, however, no studies attempting to elucidate the different incidences of 11q23/MLL rearrangements detected by the two methods in specific subgroups of leukemia.

In the present study of 51 infants with acute leukemia, we found 13 patients in whom cytogenetic analysis and Southern blot analysis using an MLL cDNA probe resulted in contradictory findings. To elucidate the different incidences of 11q23/MLL rearrangements in infant leukemia detected by the two methods, fluorescent in situ hybridization (FISH) analysis using an MLL probe was performed.

Materials and methods

Patient samples

Bone marrow (BM) or peripheral blood (PB) samples that were obtained from 51 infants (26 males and 25 females) with acute leukemia under 12 months of age (median age 6 months), who were hospitalized in 28 institutions throughout Japan, were transferred to Saitama Cancer Center by one or two overnight deliveries. These samples were collected during the period between April 1985 and March 2000. In all, 28 patients were diagnosed as having ALL and the remaining 23 patients as having AML on the basis of the FAB classification.⁸

Cytogenetic studies

Chromosomes from BM or PB cells were studied by the methods described previously.⁶ The number of metaphase cells examined ranged from four to 20 with a median of 11. Chromosome abnormalities were described according to the International System of Human Cytogenetic Nomenclature (ISCN) 1995.⁹ Karyotypes from 17 patients with AML were published elsewhere.⁶

Southern blot analysis

DNA was digested with BamHI and/or HindIII, electrophoresed in 0.8% agarose gels, and transferred onto nylon membranes (Hybond N+, Amersham, Backs, UK). Hybridization was performed with an MLL cDNA probe, probe X, which covered the breakpoint cluster region (bcr) (exons 8–14) of MLL (Figure 1a).^10,11 In one patient (No. 331), probe X and another MLL cDNA probe, pSPR25-1, which covered the 1.9 kb cDNA region containing the MLL bcr (exons 4–16), and BamHI, HindIII, EcoRI, XbaI, or BglII digestion were used.

Figure 1.

(a) A partial map of the 11q23 region including the MLL gene and its bcr. The position of the Vysis MLL probe (green and red rectangles) used for FISH and that of the MLL cDNA probe (open yellow rectangle) used for Southern blot are shown below the map. (b) FISH analysis using the Vysis MLL probe on No. 296: the green (5' part) and red (3' part) signals are split in the interphase cell, but not in the metaphase cell. (c-1) FISH analysis using the MLL probe in an apparently normal metaphase cell of No. 1477: the green and red signals of normal sizes are located in one C-group chromosome, the green signal of a smaller size is located in the short arm of another C-group chromosome, and the green signal of an intermediate size and the red signal of a smaller size are located in the other C-group chromosome. (c-2) Metaphase FISH analysis using the MLL probe and a chromosome 10 painting probe (red) on No. 1477: the green signal of a smaller size is located in the short arm of chromosome 10. (c-3) Metaphase FISH analysis using the MLL probe and a BAC probe targeting AF10 (red) in No. 1477: the green (5'-part MLL) and red (AF10) fusion signal is seen in der(10) chromosome. The red MLL signal of a smaller size was visible in the other C-group chromosome of the c-2 and c-3 metaphase cells under the microscope; however, the red MLL signals on the C-group chromosome artificially disappeared when the images were photographed. (d-1) Metaphase FISH analysis using the MLL probe in No. 2054: the green signal is seen in the long arm of one B-group chromosome, the red signal in one C-group chromosome, and the green and red signals in another C-group chromosome. (d-2) Metaphase FISH analysis using the MLL probe and a chromosome 4 painting probe (red) in No. 2054: the green MLL signal is seen in chromosome 4. (e-1) Metaphase FISH analysis using the MLL probe in No. 2134: the green signal is seen in der(11)t(1;11) chromosome and the red signal in the marker chromosome. (e-2) G-banded partial karyotype (the lower row) showing a marker chromosome and der(11)t(1;11)(q21;q13), and the same chromosomes analyzed by spectral karyotyping (the upper row) or by the Rx-FISH method (the middle row) of No. 2134. (f) Metaphase FISH analysis using the MLL probe (green and red), the chromosome 19 painting probe (green), and the chromosome 11 centromere probe (red) in No. 1494: the red MLL signal is seen in chromosome 19, and a part of chromosome 19 (green) is seen in chromosome 11 indicating the presence of a reciprocal 11;19 translocation. The green MLL signal and green chromosome 19 painting signal are overlapped in der(11)t(11;19) chromosome. The chromosome 19 material (green) is seen on der(6)t(6;19) and der(19)t(6;19) chromosomes indicating the presence of t(6;19). (g) FISH analysis using the MLL probe in No. 2060: the green and red signals are not separated in either metaphase or interphase cells. (h) Interphase FISH analysis using the MLL probe in No. 331: the green and red signals are separated, although Southern blot using two MLL cDNA probes showed the germline MLL bands (not shown).

Full figure and legend (490K)

FISH and probes

Samples that were used in to cytogenetic studies were also used for FISH analysis. Probes used included a Vysis MLL probe (Vysis, Downers Grove, IL, USA) covering the MLL locus on chromosome 11q23, a BAC probe (Rp11-177-h22) covering the AF10 locus on chromosome 10p12, a BAC probe (ATCC 65438) covering the centromeric region of chromosome 11, and whole painting probes for chromosome 4 (Vys-120004), 10 (Vys-120010) or 19 (Vys-120019). The MLL probe generates a rearranged signal pattern by a separation of two differently labeled DNA clones, one green (proximal, 5'-, 350 kb) and one red (distal, 3'-, 190 kb) signals, located on either side of the MLL breakpoint cluster region (Figure 1a). The AF10 and 11 centromere probes were labeled with digoxigenin-11-dUTP by nick translation. A digoxigenin-labeled painting probe for chromosome 4 or 10, and a biotin-labeled painting probe for chromosome 19 were also used. Two-color FISH was performed as described previously.¹² In all, 200 interphase cells were analyzed with a cutoff value of 1.5% for false positive.

Rx-FISH and spectral karyotyping

We performed Rx-FISH in one patient (No. 2134) according to the manufacturer's protocol using an Rx-FISH color chromosome analysis FISH kit (Applied Imaging, UK). The images were captured and analyzed using an Applied Imaging Chromofluor system (Applied Imaging). Spectral karyotyping was also performed in the same patient (No. 2134) using commercial probes purchased from Applied Spectral Imaging (Carlsbad, CA, USA).

RT-PCR analysis

RNA was extracted from frozen BM samples or fixed cells in methanol and acetic acid solution according to the acid guanidinium thiocyanate/phenol/chloroform method.¹³ A first-strand cDNA was synthesized and PCR was performed with primers: MLL-4017 from MLL ex9 and AF-211r for MLL–AF4 transcripts,¹⁴ ALL1-5 from MLL ex8 and VT15-7 for MLL–AF1q transcripts, AF1Q253 and MLL ex10A for AF1q–MLL transcripts,¹⁵ MLL-4017 from MLL ex9 and AF10 pmr 29 or AF10 pmr 18 for MLL–AF10 transcripts,¹⁶ 3759U from MLL ex7 and 247L from ELL for MLL–ELL transcripts,¹⁷ 3759U from MLL ex7 and 216L from ENL for MLL–ENL transcripts,¹⁷ and 3.2c from MLL ex3 and 6.1 from MLL ex9 for MLL partial tandem duplication transcripts.¹⁸ RT-PCR products were purified and directly sequenced on a sequencer (Applied Biosystems, Foster City, CA, USA).

Results

In total, 51 samples were classified into four groups on the basis of chromosome changes and the MLL status (Table 1). Twenty samples showed rearranged MLL and 11q23 translocations (group A, 39%), 11 showed rearranged MLL and a normal karyotype or chromosome abnormalities other than 11q23 translocations (group B, 22%), two had germline MLL and 11q23 translocations (group C, 4%), and 18 showed germline MLL and normal karyotype or chromosome abnormalities other than 11q23 translocations (group D, 35%).

Table 1 - Chromosome changes and the MLL status in 51 infants with acute leukemia.

Full table

Clinical, cytogenetic, and molecular-genetic findings of groups B and C are summarized in Table 2. Of 11 group B patients (MLL-R/no 11q23t), seven had normal karyotype, two (identical twins) had der(11)t(1;11)(q21;q13),+mar, one had t(6;19)(q21;p13), and one had +6,add(17)(q21). Of the seven patients with normal karyotype, five had separation of MLL signals in interphase cells and no separation of the signals in metaphase cells (Nos. 296, 942, 1856, 2151, and 2155) (Figure 1b), and two had separation of MLL signals in both interphase and metaphase cells (Nos. 1477 and 2054). The FISH findings of the five patients indicated that leukemic cells were in the nondividing phase and that normal dividing cells were karyotyped. The subsequent RT–PCR using the MLL and AF4 primers detected no PCR product in all the five samples.

Table 2 - Clinical findings, karyotypes and the MLL status in 13 acute leukemia infants with discrepancy between cytogenetic and Southern blot findings.

Full table

Since the green signal (5' part of MLL) was located in the short arm of a C-group chromosome in No. 1477, insertion of the 5' MLL fragment into the short arm of chromosome 10 was suspected (Figure 1c-1). FISH with a WCP probe identified that the green MLL signal was present in the short arm of chromosome 10 (Figure 1c-2), and subsequent FISH analysis using the MLL and AF10 probes detected the MLL–AF10 fusion signal on chromosome arm 10p (Figure 1c-3). Thus, cryptic insertion of the MLL into the AF10 locus was confirmed in No. 1477. Since the green MLL signal on chromosome 10 was smaller than that on der(11) chromosome, and the red MLL signal on der(11) chromosome was smaller than that on chromosome 11 (Figure 1c-1), the 5' MLL fragment inserted into the AF10 locus may be less than 200 kb in size, and the MLL fragment downstream of the breakpoint appeared to be partially deleted. RT-PCR failed to detect the MLL–AF10 fusion transcript in the leukemic cells, probably because the primers used were inappropriate. Likewise, the green signal located in the proximal part of the long arm of a B-group chromosome in No. 2054 suggested insertion of the MLL fragment in the long arm of chromosome 4 (Figure 1d-1). FISH with a WCP probe identified that the MLL signal was present in the long arm of chromosome 4 (Figure 1d-2), and subsequent RT-PCR detected the MLL–AF4 fusion transcript in the leukemic cells. These findings showed cryptic insertion of the MLL fragment into the AF4 locus in No. 2054. Since no green signal was seen on der(11) chromosome, the inserted fragment may be greater than 350 kb in size.

Of four patients with rearranged MLL and an abnormal karyotype with no 11q23 translocation, two identical twins (Nos. 2134 and M201) had the same MLL rearrangements and the same abnormal karyotype, suggesting the monoclonal origin of leukemic cells of the twins. FISH detected the green MLL signal on the proximal part of the long arm of der(11) chromosome, and the red MLL signal on the marker chromosome (Figure 1e-1). Spectral karyotyping analysis showed that the der(11) and marker chromosomes consisted of chromosomes 1 and 11, and showed that two fragments from chromosome 11 were separately inserted into the marker chromosome (Figure 1e-2, the upper karyotype). Rx-FISH analysis showed that the terminal portion of the long arm of the marker chromosome was derived from the terminal 1p fragment (Figure 1e-2, the middle karyotype). Subsequent RT-PCR analysis identified the MLL–AF1q fusion transcript. These findings suggest that the reciprocal translocation t(1;11)(q21;q23) occurred first, followed by the complex translocation involving four breakpoints at 1p32, 1p12, 11q13, and 11q23. Thus, the der(11)t(1;11)(q21;q13) chromosome may be revised as der(11)t(1;11)(11pter11q13::11q2311q23::1q211qter), and the marker chromosome may be described as der(1)t(1;11)(1p121q21::11q2311q25::1p121p32::11q1311q23::1p321pter). Thus, cryptic MLL–AF1q rearrangements were identified.

In one patient (No. 1494) with rearranged MLL and t(6;19)(q21;p13), FISH with the chromosome 19 painting probe, the 11cen probe, and the MLL probes detected the presence of the 3' part of MLL on apparently normal chromosome 19, but not on der(6) or der(19) chromosome, and that a part of chromosome 19 was translocated to chromosome 11 (Figure 1f). An additional FISH analysis detected the presence of the 5' part of MLL and absence of the 3' part of MLL on apparently normal chromosome 11 (der(11)t(11;19)(q23;p13)). The karyotype was revised as 46,XX,t(6;19)(q21;p13), t(11;19)(q23;p13) after FISH analysis. RT-PCR analysis failed to detect either the MLL–ELL or MLL–ENL fusion transcript in the leukemic cells. FISH analysis on one patient (No. 1982) with rearranged MLL and +6,add(17)(q21) showed no separation of the MLL signals in both interphase and metaphase cells, suggesting the presence of a partial tandem duplication of MLL. However, RT-PCR analysis using the MLL primers failed to detect the PCR products, which are expressed in acute leukemia with the partial tandem duplication of MLL.

One (No. 2060) of two group C patients (MLL-G/11q23t) showed t(11;19)(q23;q13). FISH analysis detected no separation of the MLL signals in either metaphase or interphase cells (Figure 1g), suggesting that the breakpoint of the 11q23 translocation is located distal to the MLL locus. The other group C patient (No. 331) showed 14 metaphase cells with t(4;11) by cytogenetic analysis. FISH analysis detected separation of the MLL signals in interphase cells (Figure 1h) and no abnormal metaphase cells were found at the time of FISH analysis. Southern blot analysis using another MLL cDNA probe failed to detect rearranged bands, and RT-PCR analysis also failed to detect the MLL–AF4 fusion transcript. These findings suggest that the breakpoint may be located outside the MLL bcr region and inside the MLL locus in this patient.

Discussion

Infant acute leukemia is specifically associated with 11q23 translocations that fuse the MLL gene at 11q23 with at least 50 partner genes at various chromosome bands.^4,6,7 The 11q23/MLL rearrangements are also found in many patients with AML-M4/M5 regardless of age.³ In the present study of 51 infants with acute leukemia, cytogenetic findings and Southern blot findings using an MLL cDNA probe were contradictory in 13 (25%). One previous study of 79 ALL infants whose karyotype and MLL status were successfully studied reported contradictory findings in 17 (22%): rearranged MLL and normal karyotype in eight (8/79, 10%), rearranged MLL and abnormal karyotype with no 11q23 translocation in seven (9%), and germline MLL and 11q23 translocations in two (3%).⁴ Another study of 47 ALL infants whose karyotypes were successfully studied reported rearranged MLL and normal karyotype in three (3/47, 6%).¹⁹ In the other study of 14 adult patients with AML-M4/M5 and MLL rearrangements, six had normal karyotype and two had abnormal karyotype with no 11q23 translocation.³ Thus, the present and the previous studies showed that the discrepancies in the incidences of 11q23/MLL rearrangement between cytogenetic and molecular genetic findings are not uncommon in infant leukemia and in adult AML-M4/M5.

FISH analysis of seven patients with rearranged MLL and normal karyotype disclosed the presence of normal dividing cells and nondividing leukemic cells in the same BM in five, and cryptic insertion of the 5'-MLL fragment into 10p or 4q in two. In a study of 3 patients with acute promyelocytic leukemia with the PML–RARA fusion transcript detected by RT-PCR and normal karyotype, FISH disclosed the PML–RARA fusion signal only in interphase cells in one and cryptic insertion of the RARA fragment into the PML locus in two.⁵ Thus, normal karyotype could be interpreted by one of the three instances: (1) the karyotype of normal hematopoietic cells was analyzed and leukemic cells were in the nondividing phase, (2) the karyotype appeared to be normal, but cryptic insertion or translocation that is detectable by FISH using appropriate probes may be present, and (3) the karyotype of leukemic cells was shown to be normal by the cytogenetic and FISH methods.

The present FISH and RT-PCR study also disclosed cryptic MLL translocation in three of four patients with rearranged MLL and abnormal karyotype with no 11q23 translocation. Thus, abnormal karyotype without 11q23 translocation does not necessarily mean that the primary genetic event such as the MLL fusion is not present in leukemic cells.

The present two patients had cryptic insertion of the MLL fragment into the AF10 locus on chromosome arm 10p, or into the AF4 locus on 4q. Since the introduction of FISH analysis, there have been an increasing number of reports on cryptic insertion in specific subtypes of leukemia and solid tumor.^{20,21,22,23,24} Cryptic insertions were classified into two types: one has the opposite transcriptional orientations of the two genes forming a fusion in relation to the centromere, and the other has the same orientation of the two genes in relation to the centromere.^{20,21,22,23,24} The inv ins(10;11)(p12;q23q23) found in the present patient (no. 1477) belongs to the former type, and the inverted insertion produced a fusion of the MLL and AF10 genes that are transcribed oppositely in relation to the centromere in the normal chromosome 10 and 11 locus. In this type of insertion, at least three chromosome breaks and an inversely oriented insertion of one of the genes into the other are required to generate a functional fusion gene. When the inserted segment is small, the insertion is undetectable by cytogenetic analysis, and is called cryptic. An inverted insertion of the larger MLL fragment into the AF10 locus that was detected by cytogenetic analysis was reported previously in an adult patient with AML-M5a.²⁵ Other examples of the cryptic inverted insertion are inv ins(21;22)(q12;q12q12) forming the EWS–ERG fusion reported in a Ewing sarcoma,²⁰ and inv ins(9;12)(q34;p12p12) forming the ETV6–ABL fusion reported in chronic myeloid leukemia.²²

One patient (No. 2054) with ins(4;11)(q21;q23q23) belongs to the other type. The insertion produced a fusion of the MLL and AF4 genes that are transcribed in the same orientation in relation to the centromere. Recently, Von Bergh et al²⁴ reported a similar insertion of the 5' MLL fragment into the AF4 locus in an adult patient with ALL. Using various PAC clones, they evaluated the size of the inserted fragment as approximately 250 kb that is unidentifiable by cytogenetic analysis, and the insertion is reasonably called cryptic. A cryptic insertion of the AML1 fragment into the ETO locus, or the insertion of the ETO fragment into the AML1 locus was reported in two patients with AML.²³ Similar cryptic insertions forming the PML–RARA fusion gene or the BCR–ABL fusion gene were reported in AML-M3 or in chronic myeloid leukemia.^5,20 Thus, cryptic insertions forming the fusion of the genes that are transcribed in the opposite orientation or in the same orientation in relation to the centromere appear to be a recurrent mechanism for the neoplastic process in leukemia and solid tumor.

The same MLL rearrangements identified by Southern blot analysis and the same complex translocation involving chromosomes 1 and 11 were found in the two twin patients in the present study. The plausible explanation for the findings is that the MLL–AF1q fusion gene occurred in one blood stem cell of one twin in utero.

Subsequently, descendent progeny of this transformed cell spread to the other twin via the placenta.²⁶ In total, 10 pairs of monozygotic twins including the present ones showed the same MLL rearrangements and/or the same 11q23 translocations, and their median age was 3 months old.²⁷ Four pairs of monozygotic twins showed the same ETV6–AML1 rearrangements, and their median age was 4 years.²⁸ The different median ages of the two groups of leukemia twins may be explained by the hypothesis that transformed cells with the MLL fusion gene have greater growth potential and require fewer genetic events to full-blown leukemic cells than the cells with the ETV6–AML1 fusion gene.

In conclusion, the present FISH study clarified why cytogenetic studies and molecular studies using Southern blot or RT-PCR sometimes resulted in contradictory findings. The presence of normal dividing cells and nondividing leukemic cells in the same bone marrow, and cryptic insertion or translocation caused the discrepancy. FISH should be used to complement cytogenetic analysis when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.⁶

References

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Acknowledgements

This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health, Welfare and Labor of Japan. We thank Dr M Seto for providing MLL cDNA probes, probe X and pSPR25-1, and Drs Y Arai and M Ohki, National Cancer Center Research Institute in Japan for providing a BAC probe (Rp11-177-h22).