Therapy and Follow up Studies

Use of dual-color interphase FISH for the detection of inv(16) in acute myeloid leukemia at diagnosis, relapse and during follow-up: a study of 23 patients

Abstract

The value of dual-color fluorescence in situ hybridization (FISH) for the detection of inv(16), using two contigs of cosmid probes mapping on both sides of the chromosome 16p breakpoint region, was evaluated in 23 acute myeloid leukemias (AML) in different phases of the disease. At diagnosis interphase FISH detected inv(16) in 19/19 (100%) cases with conventional cytogenetics (CC) evident aberration and excluded the rearrangement in two patients with CC suspected inv(16). Moreover, it also identified an associated del(16p) in two patients. At relapse, it revealed the inv(16) in 8/8 (100%) studied cases. These results were concordant with those of reverse transcriptase-polymerase chain reaction (RT-PCR). From 13 patients who obtained at least one complete remission (CR), 31 follow-up samples were analyzed using interphase FISH. Twenty-nine specimens scored negative for inv(16) and two were positive. RT-PCR detected CBFβ/MYH11 transcripts in four of the nine CR samples analyzed, being more sensitive than interphase FISH. Eight of the 13 patients relapsed at a median time of 6.5 months (range 1–15) from the last negative FISH analysis. Of the two patients with positive FISH in CR, one relapsed soon after. At diagnosis and relapse, interphase-FISH proved to be an effective technique for detecting inv(16) appearing more sensitive than CC. Prospective studies with more frequent controls and possibly additional FISH probes are needed to assess the value of interphase FISH for minimal residual disease (MRD) and relapse prediction.

Introduction

The inv(16)(p13q22) or the related t(16;16)(p13;q22) is a chromosomal aberration observed in approximately 10% of de novo acute myeloid leukemia (AML), mostly classified as French–American–British (FAB) M4Eo subtype. This subset is characterized by myeloblastic/monoblastic infiltration of bone marrow (BM), elevated monocyte count in peripheral blood and presence of abnormal BM eosinophils accounting for more than 5% of the non-erythroid cells.12 At the molecular level, this rearrangement disrupts the heavy chain of smooth muscle myosin (MYH11) gene at 16p13 and the core binding factor beta (CBFβ) at 16q22, leading to the production of a specific chimeric gene (CBFβ/MYH11).34

Several studies have suggested that the presence of inv(16) is an important prognostic indicator in AML patients, as it is associated with a relatively favorable outcome: 90% of patients obtain CR, with nearly 50% being disease-free survivors at 5 years.56

The inv(16) may be difficult to be detected with CC, particularly when poor quality metaphases are obtained from leukemic cells. Moreover, CC cannot detect cryptic deletions of sequences centromeric to the p-arm breakpoint which have been described in a subset of inv(16) AML patients, accounting for 18–33% of cases.78910 The RT-PCR is able to detect CBFβ/MYH11 fusion transcript in the majority of inv(16)-positive cases at diagnosis; however, some cases remain RT-PCR negative. This finding might be caused by the diversity among inv(16) chimeric transcripts, together with the presence of alternatively spliced transcripts or poor quality RNA.1112

FISH technique, using genomic probes spanning the breakpoint region, can detect structural rearrangements both in leukemia metaphase and interphase cells.1314 Because interphase FISH does not require cell division, it can be applied for quantitative evaluation of MRD during or after therapy.

FISH probes, previously reported to detect inv(16) are the cosmids C36 and C40, suitable only for metaphases15 and a yeast artificial chromosome (YAC) probe, spanning the 16p breakpoint.16 Although applicable on interphase cells, the YAC gives false negative results in cases of concomitant 16p deletion.16

In the present study we analyzed 23 AML patients with CC clear or suspected inv(16), using a new set of cosmid contigs that allows interphase analysis,8 with the aim of evaluating the efficacy of interphase dual-color FISH in detecting inv(16) at the onset of the disease, through its course and at relapse. Results were compared with CC and RT-PCR analysis of CBFβ/MYH11 transcript.

Materials and methods

Patients

BM specimens were collected from 23 patients with a cytogenetically clear or suspected inv(16) AML (12 males and 11 females, median age 33, range 5–74) diagnosed and treated, between 1991 and 1997, in four different Centers (Center for Human Genetics, University of Leuven, Belgium (cases 1 to 10); Department of Cellular Biotechnologies and Hematology, University ‘La Sapienza’ of Rome, Italy (cases 11 to 19); Hematology and Bone Marrow Transplantation Unit, University of Perugia, Italy (cases 20 to 22); Hematology Civil Hospital, Avellino, Italy (case number 23). The inclusion of the patients was not consecutive, but based on the availability of sequential samples and/or a history of clinical relapse.

BM samples from five patients with thrombocytopenia and a normal karyotype were used to determine the frequency of interphase cells with an apparent inv(16) in normal controls and to establish the cut-off level above which a diagnosis of inv(16) could be done.

Cytogenetics

Cytogenetic analyses of the BM cells were performed using standard methods. The chromosomes were identified by R and G bands and classified according to the ISCN (1995).17

Dual-color fish

Two cosmid contigs (each approximately of 100 kb), cloned by Dauwerse et al8 and mapping to either side of the p-arm breakpoint region of chromosome 16 were used as probes. The proximal contig consisted of cosmids zit 14, zit 18, zit 38. The distal contig consisted of the cosmid zit 27, zit 29 and zit 80.

Slides prepared for cytogenetic analysis were used for interphase and metaphase FISH. Before hybridization, the slides were pre-treated with RNA-ase and pepsin, followed by fixation and denaturation. Dual-color FISH was performed as previously reported.18

Briefly, the probes were labeled by nick-translation with biotin-16-dUTP (cosmids zit 27, zit 29 and zit 80) or digoxygenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany) (cosmids zit 14, zit 18 and zit 38). The probes (5 ng/μl, each) were denatured in 70% formamide at 72°C, preannealed with 200 × excess Cot-1 DNA for 45 min at 37°C and hybridized overnight. After removal of the excess of probes, hybridization signals were visualized by immunofluorescence using avidin-FITC and Texas red-conjugated antibodies. Nuclei and chromosomes were counter-stained with DAPI. For each sample, at least 300 nuclei were scored using a fluorescent microscope equipped with a triple band pass filter for simultaneous excitation of FITC, Texas red and DAPI. All the FISH analyses were done by two investigators (CM and EB), who were blind to the cytogenetic and RT-PCR results.

RT-PCR

RT-PCR detection of CBFβ/MYH11 transcripts was performed on available diagnostic and follow-up samples.

Total RNA was extracted from Ficoll–Hypaque isolated mononuclear cells using the Chomczynski and Sacchi method.19 The quality of RNA was checked by running 1 μg into an agarose gel containing formaldehyde. The reverse transcription was performed using 1 μg of total RNA in a 20 μl volume at 42°C for 40 min, followed by incubation at 99°C for 5 min in a mixture containing random hexamers as primers, 1 × PCR buffer (500 mM KCl, 100 mM Tris-Cl pH 8.3), 5 mM MgCl2, 1 mM dNTPs, 1 U RNAsin and 2.5 U MuLV RT according to the instructions of the manufacturer (RNA PCR core kit; Perkin-Elmer, Hoffmann-La Roche Inc, USA). A volume of 5 μl of the cDNA was diluted in 45 μl of a mixture containing 1 × PCR buffer (the same used for the RT step), 2.5 mM MgCl2, 200 μM of each dNTPs, 6% deionized formamide, 20 pmol of each primer (C1, M1, M3 see below) and 2.5 U of TAQ Gold DNA Polymerase.

The sequence of the primers used in the reaction were: C1 sense 5′ GCA GGC AAG GTA TAT TTG AAG G 3′ (nt 253–274 of CBFβ); M1 antisense 5′ TCC TCT TCT CCT CAT TCT GCT C 3′ (reverse sequence of nt 2095–2116 of MYH11); M3 antisense 5′ TGA AGC AAC TCC TGG GTG TC 3′ (reverse sequence of nt 1316–1297 of MYH11).

After an initial denaturation at 95°C for 9 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 45 s were performed using the 2400 thermal cycler (Perkin-Elmer) for a total of 45 cycles. At the end of the reaction 15 μl were run on a 1% agarose gel stained with ethidium bromide and visualized under a UV lamp.

To test the efficiency of the RT step normal PBGD (Porfobilinogen deaminase) cDNA was amplified in each experiment at the same conditions used above, with the following primers: PBGD sense 5′ CTG GTA ACG GCA ATG CGG CT 3′ (nt 32–51); PBGD antisense 5′ GCA GAT GGC TCC GAT GGT GA 3′ (reverse sequences of nt 350–369).

To assess the sensitivity of the RT-PCR assay, total RNA, isolated from a PCR-positive patient with 90% of BM blast cells, was serially diluted with total RNA extracted from normal PB mononuclear cells. PCR was performed at the same conditions and cycling parameters above mentioned.

The patients from Leuven were studied using the same primers and almost the same conditions.20

Results

Clinical data

Twenty-three AML patients were included in the study. Diagnoses according to the FAB classification were: M2 (two cases), M2Eo (one case), M4 (three cases), M4Eo (13 cases), M5 (three cases), and M5a (one case) acute leukemias. Most patients received intensive chemotherapy and, for all but five, CR was obtained. Autologous or allogeneic BMT was given to 12 patients either before or after relapse. Relapses occurred in 15 patients at a median time of 11 months (range 5–24) from CR. At present, five patients are alive in first CR (CR1), five in second CR (CR2), one in third CR (CR3) while 12 patients have died. The clinical history of patient No. 3 was previously reported.21

Cytogenetics

Cytogenetic results are detailed in Table 1. Nineteen diagnostic and nine relapse samples investigated were inv(16) positive. Additional changes were seen at diagnosis in four cases, namely +8 (No. 16), +21 (No. 20), +22 (Nos 1 and 7) and at relapse in two cases, namely +8 (No. 3) and +9 (No. 11). Two diagnostic marrows (Nos 18 and 19) showed a suspected inv(16) in a minority of cells.

Table 1  Results of cytogenetics, interphase FISH and RT-PCR detection of inv(16) in AML

In CR, 28/30 BM showed a normal karyotype; in two cases, one or two cells with suspected inv(16) were seen. In the PR case (No. 9), inv(16) was documented in three of the 30 analyzed cells.

In situ hybridization

The proximal cosmid contig (zit 14, 18, 38) gives a red signal and the distal cosmid contig (zit 27, 29 and 80) a green signal. On normal chromosome 16 the red and the green spots colocalize on p-arm showing fused or contiguous red and green signals while, on the inverted chromosome 16, they appear as distinct and distant spots: the red one on the q-arm and the green one on the p-arm. In interphase nuclei, three patterns could be observed: (1) nuclei with two colocalized or fused red and green spots, scored as inv(16) negative; (2) nuclei with one red and green fused spot and one red and one green independent spots, scored as inv(16)-positive cells (Figure 1); (3) nuclei with the loss of red fluorescent signal in case of inv(16) with a small deletion proximal to the 16p breakpoint (Figure 2).

Figure 1
figure1

 Nucleus with inv(16) (case No. 12): the presence of the rearrangement is indicated by separate red and green signals.

Figure 2
figure2

 Nuclei with inv(16) and del(16p) (case No. 13): the coexistence of both rearrangements is indicated by the loss of the separated red spot.

Hybridization experiments on five BM controls showed a mean percentage of 1% (range 0.5–1.5%) of cells with one red and one green separate signal, with a standard deviation of 0.5%. The cut-off value for positive nuclei (mean ±3 standard deviations) was set-up at 2.5. This value represents the technical limit for the detection of MRD using these contigs probes.

Patients

FISH results are given in Table 1 together with RT-PCR data. Nineteen cases studied at diagnosis were clearly positive with a median percentage of positive nuclei of 81% (range 60–95%). The two patients (Nos 18 and 19) with a suspected inv(16) by CC, tested negative by interphase FISH (rate of positive nuclei of 1 and 2%, respectively) and by RT-PCR. Nine cases studied at relapse showed clear evidence of inv(16) (median 67.5%, range 26–88), results that were concordant with CC and RT-PCR.

In two patients (No. 4 and No. 13) deletion of sequences proximal to the inv(16) p-arm breakpoint, was identified by the loss of the red signal (proximal contig).

Thirty-one follow-up samples from 13 CR patients and one PR sample were studied between 1 and 67 months after diagnosis. The scores were clearly below the cut-off value (2.5%) in 28 instances; three follow-up BM showed a positive score: (1) case No. 9 had 13% of positive nuclei, while in PR after 1 month of therapy; (2) case No. 1, a patient in CR2 67 months after diagnosis, had 10% of positive nuclei. This patient also showed by CC two out of 10 metaphases with dubious inv(16); clinically, he was and remained in CR until the last follow-up, 7 years after diagnosis; (3) case No. 10 had 3.5% of positive nuclei, while in CR, 4 months after diagnosis and 1 month before clinical relapse. This case, although still in CR1, also had one out of 25 metaphases with a dubious inv(16) by CC (Table 1). Altogether, eight patients relapsed at a median time of 6.5 months (range 1–15) from the last negative FISH analysis.

RT-PCR

RT-PCR showed the presence of CBFβ/MYH11 fusion gene transcripts in 10 cases at diagnosis and three cases at relapse. The two cases negative at diagnosis by interphase FISH were also negative by RT-PCR. Nine CR samples were investigated for the evaluation of MRD: four samples were positive, and five were negative.

Three of the four positive samples were negative by interphase FISH indicating the higher sensitivity of the RT-PCR assay. Sensitivity analysis showed that the RT-PCR assay could detect 10 pg of rearranged RNA in 1 μg of normal RNA.

Discussion

The aim of the study was to evaluate the reliability of dual-color interphase FISH to identify inv(16)(p13q22). Twenty-three non-consecutive AML patients, with a clear or a suspected inv(16) by CC, were analyzed by interphase FISH at diagnosis, relapse and CR, altogether 61 samples (Table 1).

For the identification of inv(16), probes mapping on both sides of the 16p breakpoint were chosen; the presence of repetitive DNA in the 16p breakpoint region, cross-hybridizing with the 16q DNA regions, limited the choice of genomic probes, thus we used contig probes laying at some genomic distance one from the other. This explains why the signals appear more often contiguous than truly overlapping and why in a small number of cells they are seen as noncontiguous. As a consequence, the cut-off to exclude false positive cells was established at 2.5%.

The probes used in this study proved to be very specific: all samples with inv(16) by CC were identified by interphase FISH, with largely positive score; furthermore, inv(16) cases masked by translocation were detected in a previously reported study.20 These probes could also demonstrate the rearrangement in rare cases not detected by RT-PCR because of an unusual breakpoint.11 More importantly, uncertain cytogenetic results caused by absent or poor quality metaphases will be clarified, beyond doubt.22

As seen in two patients, these two-color FISH assays also identified the cryptic del(16p), previously reported to have an incidence between 18 and 33% of inv(16) cases.910 In our series, however, the incidence of del(16p) was lower (9%) than that reported, but this may be explained by patient selection, mainly based on the occurrence of a clinical relapse.

Thirty-one follow-up samples (30 in CR, one in PR) were analyzed to evaluate the sensitivity of interphase FISH and its possible use for prediction of relapse. Only three samples scored positive and, of these latter, only one relapsed shortly after. In the other eight relapsed cases, no evidence of MRD was obtained in CR. This result might be related to a low sensitivity of interphase FISH (cut-off 2.5%) and/or to a too extended time interval between BM controls performed during CR. In fact, the kinetics of AMLs could be such that it is not reasonable to expect a measurable number of residual leukemic cells months before relapse.

Other probes have been proposed for the FISH detection of inv(16), all mapping on the 16p breakpoint region. These are the cos 36 and cos 40 which are excellent tools on metaphases but genomically too distant, so that they do not reliably colocalize in interphase cells.15 Furthermore, the proximal probe lies outside and thus does not detect the associated 16p deletion.20 A YAC covering the breakpoint region and split by inv16 is also available and has been tested in some of the cases studied (data not shown). The YAC gives two spots on normal nuclei and three spots in cells with inv(16). However, occasional trisomy 16 or cross-hybridization with 16q sequences cause a rather high false positive rate while, on the other hand, in the case of associated 16p deletion, the loss of one signal results in a false negative diagnosis.16

RT-PCR detection of CBFβ/MYH11 transcript could only be performed in a limited number of samples. At diagnosis and relapse RT-PCR and interphase FISH results were concordant. As expected, RT-PCR was more sensitive than CC or interphase FISH and indeed was found positive in three CR samples, negative with the other techniques. Two of these cases had a clinical relapse 3 months later, indicating that a positive result by RT-PCR analysis per se is not necessarily predictive of impending relapse: in t(8;21) leukemia, for example, the result of RT-PCR obtained in CR may remain positive even after years of remission.23

In conclusion, the two-color FISH assay for inv(16) is specific and constitutes an excellent tool to be used at diagnosis (and relapse) of AML on all occasions where the presence of inv(16) is questioned or ambiguous. For MRD, there is still a need for improvement of inv(16) detection methods. Interphase FISH can be combined with cell sorting of specific immunophenotype; alternatively, use of additional probes with further colors in order to better visualize both 16p and 16q breakpoints can reduce the false positive rate. RT-PCR appears more sensitive, but quantitative methods should be developed and a good study on the value of RT-PCR in prospective longitudinal follow-ups of inv(16) acute leukemias is still lacking.

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Acknowledgements

This work was supported by Biomed Concerted Action: contract grant CT 94-1703; the Belgian programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming (the scientific responsibility is assumed by the authors); the Flemish Government in the frame of the action Kom op tegen Kanker/Vlaamse Kankerliga and partially by MURST fondi 60% (GA). We gratefully acknowledge the collaboration of Dr A Bosly, P Brock, A Ferrant, D Selleslag and G Verhoef, for providing the clinical data and of Dr B Santulli for providing cytogenetic data of patient No. 23. C Mecucci is partially supported by AIRC.

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Mancini, M., Cedrone, M., Diverio, D. et al. Use of dual-color interphase FISH for the detection of inv(16) in acute myeloid leukemia at diagnosis, relapse and during follow-up: a study of 23 patients. Leukemia 14, 364–368 (2000). https://doi.org/10.1038/sj.leu.2401678

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Keywords

  • inv(16)(p13q22)
  • acute myeloid leukemia
  • FISH
  • conventional cytogenetics
  • RT-PCR

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