To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: −5, −7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53deletion or trisomy 8 (one case each) in 33–60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76–94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.
The identification of chromosome aberrations in acute myeloid leukemia (AML) has importance in clinical practice1,2,3 and the efficiency of different methods for the detection of balanced and unbalanced chromosome lesions has been investigated in some studies using conventional cytogenetic analysis (CCA) and molecular cytogenetic investigations.4,5,6,7,8,9
There is evidence that some AMLs with an apparently normal karyotype may be shown to carry aneuploidy by interphase cytogenetics,10,11 and that cryptic chromosome translocations may be detected in some of these patients by molecular genetic analysis,12,13 whereas little information is presently available on the occurrence of cryptic deletions.
Because the size of a specific chromosome deletion may vary from the loss of a large portion of chromosome material to a barely detectable deletion of a sub-band, including few megabases of DNA,14 it is reasonable to assume that sub-microscopic deletions may exist that are beyond the resolution power of CCA. These small deletions may be detectable by the more sensitive fluorescence in situ hybridization (FISH) technique, capable of analyzing DNA losses in the order of a few hundred kb magnitude. When used in interphase cells FISH analysis may also enable detection of minor abnormal clones, which may sometimes be difficult to detect by CCA due to their low mitotic index.6,15
Indeed, there are some recent reports in the literature, supporting the existence of occult chromosome deletions and trisomies in some patients with myeloid neoplasias bearing a normal karyotype,16,17,18,19 but scant information is available on AML.20
We carried out a FISH study of 82 cytogenetically normal AMLs with the the following aims: (1) to determine the frequency of occult chromosome deletions and trisomies; (2) to sort out whether the failure of CCA to detect a chromosome anomaly derived from a low mitotic index of the aberrant clone or by the sub-microscopic nature of the rearrangement; (3) to identify the cell-lineage involved by the occult chromosome anomaly; and (4) to describe salient hematologic and clinical features of patients with cryptic deletions or trisomies.
Materials and methods
Two groups of cytogenetically normal AML patients were studied: group A, including 55 patients with de novo AMLs (AML-M3 excluded) in the 15–60 years age range and group B, including 21 elderly AML cases (age 62–80 years) and six AMLs developed following an antecedent myelodysplastic syndrome (AML/MDS) (age 63–78 years). The latter two entities were included in the same group because they are likely to represent biologically related forms of leukemia.21 Patients in group A were enrolled in the AML-99 GIMEMA protocol and patients in group B represented unselected cases of elderly AMLs or AML/MDS with normal karyotype seen at the Institute of Hematology, University of Ferrara between 1996 and 2000.
The diagnosis of AML was established according to the FAB criteria.22 Salient hematologic data were collected by review of clinical charts. Careful history of anamnestic exposure to myelotoxic agents was collected using a pre-determined questionnaire in those patients seen in Ferrara, as previously described.23
Cytogenetics and molecular genetics
Centralized CCA according to standard methods was performed in patients in group A on fresh bone marrow (BM) samples. These were shipped by overnight courier from peripheral GIMEMA Institutions to five referral cytogenetic laboratories (Institutes of Hematology Universities of Roma ‘La Sapienza’, Perugia, Bologna, Turin and Ferrara, Italy). Patients in group B were diagnosed and studied in Ferrara. At least 20 mitotic figures were karyotyped in all cases.
According to diagnostic guidelines approved by the centers participating in the AML-99 GIMEMA protocol, patients in group A were also routinely screened by RT-PCR and Southern blot for the following molecular aberrations: MLL breaks by Southern blotting, BCB/ABL, PML/RARA, AML1/ETO and CBFβ/MYH11 by RT-PCR. In all cases standardized methods were used for the amplification of major fusion genes associated with AML, as previously described.24
The presence of t(8;21), inv(16), t(15;17), t(9;22) or MLL rearrangement was investigated in patients in group B by FISH using commercially available probes (Vysis, Downers Grove, IL, USA, distributed in Italy by Olympus, Milan).
To detect occult chromosome anomalies, probes recognizing the following loci were used in interphase FISH analysis: 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11-12, sequences at the centromeres of chromosomes 5, 7, 8. The 7q31, 12p13, 20q11 probes and the chromosome 7, 8 centromeric probes were purchased from Vysis, from Boehringer Mannheim (Mannheim, Germany) or from Oncor (Gaithersburg, MD, USA). The 160f8 BAC probe detecting 5q10 subcentromeric sequences was prepared by M Trubia (FIMO, Milan, Italy). The PAC – P1 probe 144G9 isolated by J Landegent (Department of Hematology, AZ Leiden, The Netherlands) detecting sequences located between the IL-9 and IL-4 genes at 5q31 and a 17p13.3 cosmid recognizing p53 gene sequences were used for the detection of 5q and 17p deletions. These two probes were made available by F Birg (Institut de Cancérologie e d'Immunologie de Marseille, INSERM 119, Marseille, France) in the context of the Biomed I programme, ‘EU concerted action for cytogenetic diagnosis of hematologic malignancies’ (project leader: A Hagemeijer, Centre for Human Genetic, KUL, Leuven, Belgium).
All analyses were performed on fixed BM preparations that had been previously used for conventional karyotyping. Dual-color FISH was performed in all cases according to previously described methods,16 using a test probe and an appropriate control probe labelled with green and red fluorochromes, respectively.
At least 300 interphase nuclei counterstained with DAPI, showing well-delineated green and red signals were scored in each sample. To minimize the risk of false-positive results, signal screening was performed in those slides with high hybridization efficiency, as shown by the presence of more than 80% cells with the two expected signals of the control probe.
Based on the results of hybridization of the same probe combinations on three BM samples obtained from normal subjects aged less that 60, the cut-off point for the diagnosis of deletion and trisomy was set at the mean percentage of false positive results + 3 s.d., ie 7.5% for 5q−; 6.7% for 7q−, 8% for 12p− and 20q−, 8% for 17p−, 6% for −5 and −7; 2.5% for +8.
In addition, BM cells from 10 subjects in the 60–75 years age range were studied using the 5q31, 7q31 and 17p13 probes to rule out the occurrence of cryptic deletions involving these chromosome regions as an age-dependent phenomenon.
In 6/8 cases in which a chromosome anomaly was detected in interphase cells, 10 mitotic figures were also studied by FISH using the appropriate probe. Two cases could not be analyzed because insufficient material was available. These studies were performed to verify whether the failure of CCA to detect the chromosome anomaly derived from a low mitotic index in vitro of the abnormal clone or if, alternatively, it was due to the sub-microscopic nature of the chromosome lesion.
To sort out which cell lineages were involved by each individual chromosome anomaly a modification of the conventional FISH technique was used, permitting the direct visualization of hybridization signals on cytologically intact BM smears. The technique included four steps: (1) visualization and acquisition on a B/W camera of well spread areas on BM with preserved cell morphology; (2) hybridization procedure; (3) relocalization of previously captured areas and selection of microscopic fields with high hybridization efficiency; (4) software-assisted image capturing and attribution of pseudo-colors to enable better reproduction. The methods were described in detail previously.25
To assess the efficiency of this technique, each of the following probe combinations was tested by dual-color FISH on BM smears obtained from three subjects with non-neoplastic hemopoiesis: 5q31 and chromosome-5-subcentromeric probe, 7q31 and chromosome-7-centromeric probe, chromosome-8-centromeric probe.
Five patients with occult chromosome anomalies (two with 5q−, two with 7q−, one with +8) could be analyzed, using slides stored at room temperature for 6–18 months. Three cases were not analyzed because of technical failure (detachment of the BM film during denaturation or inefficient hybridization). The hybridization was performed according to the following protocol.
The slides were first incubated for 60 min with RNAase (100 μg/ml; Boehringer Mannheim) washed twice in 4 × SSC 5 min each, subsequently dehydrated in ethanol alcohol series (70%, 80%, 90%, 100%) and air dried. The slides were pre-warmed on a hot plate and then immersed in a 70% formamide/2 × SSC solution at 72°C for 2 min and dehydrated again with ice alcohol series. Fifteen μl of each probe were added on each slide, which was covered with a coverslip. Rubber cement was used to seal the edges and the slide was incubated overnight at 37°C, in a moist chamber. Post-hybridization washes included baths at 45°C in 50% formamide in 2 × SSC for 15 min, in 1 × SSC for 10 min and in 0.1 × SSC for 5 min, without intermittent agitation. No antifade solution was applied to the slides. Evaluation of FISH results was performed on a Nikon fluorescence-equipped microscope with couple charged camera device and appropriate hardware and software (Cytovision System, Applied Imaging distributed by Nikon, Florence, Italy). To prevent data misinterpretation deriving from inefficient hybridization those areas with more than 70% cells showing two centromeric signals at × 1200 magnification were analyzed. Blast cells, granulocytic and erythroid cell were counted as affected by 5q or 7q deletion if two centromeric signals and one 5q31/7q31 signal were present. Likewise, trisomy 8 was defined by the presence of three signals along with two signals of the control probe. Two hundred erythroblasts and granulocyte precursors were scored in the normal BM smears to set the cut-off point for recognition of 5q31, 7q31 deletion and trisomy 8. Fifty or more blast cells and a minimum of 40 erythroblasts and granulocyte precursors with well-spread nuclei were examined in each AML slide.
Cytogenetic and molecular diagnosis
More than 90% of patients enrolled in the current AML-99 GIMEMA protocol were successfully analyzed by cytogenetic and molecular genetic studies, with a 53.5% incidence of chromosomal aberrations by CCA.
Fifty-five patients included in group A had a normal karyotype in 20 or more cells. Molecular genetic investigations did not reveal major AML fusion genes or MLL rearrangements in these patients. Likewise, no cases of cryptic t(8;21), inv(16), t(15;17), t(9;22) or MLL rearrangement could be demonstrated by FISH among the 27 patients in group B.
Deletions of 5q31, 7q31, 12p13/TEL, 17p13/p53, 20q11-12 were not detected among 55 patients with adult de novo AML included in group A. Likewise, no patients in this group was found with −5, −7, +8.
As shown in Table 1, eight patients in group B (three elderly AML, five with AML following MDS) had a cryptic chromosome anomaly in 33–60% of interphase cells. A 5q31 and a 7q31 deletion were found in three cases each, a 17p13 deletion and trisomy 8 were detected in one patient each. No deletion was found in five elderly subjects affected by non-neoplastic disorders of hematopoiesis.
A sufficient number of mitotic figures could be analyzed by FISH in three patients with 5q−, in two with 7q− and in one with +8 (see Table 1). One of these patients (No. 2) had elderly AML and displayed five out of 10 metaphases with two apparently normal chromosome 5, one of which carried a submicroscopic 5q31 deletion. The remaining five patients had AML post MDS and were reported previously:16 three patients (Nos 4, 5, 7) had a submicroscopic deletion of chromosome 5q (two cases) and 7q (one case), whereas one patient (No. 6) showed two 7q31 signals on chromosomes 7 in all 15 metaphases analyzed, suggesting that the 7q− clone had a very low mitotic index in vitro. In one patient (No. 8), three out of 15 fuzzy mitotic figures showed three centromeric signals, indicating the presence of trisomy 8.
FISH on BM smears
The hybridization efficiency was found to vary in different areas of the slides. Optimal results, ie the presence of the expected two red and two green signals, were observed in well spread areas with preserved cell morphology, containing only a minority of cells with clumped chromatin. In these areas, usually representing a minority of ×1200 microscopic fields, more than 70% of the cells had the expected signal configuration.
The percentages of erythroblasts and granulocyte precursors with false 5q31/7q31 deletion or +8 in the normal BM samples was in the 4–10% range. The cut-off point for recognition of 5q deletion, 7q deletion and +8 was set at the mean values plus 2 s.d., corresponding to 12.1%, 12.5% and 10%, respectively, for erythroblasts and to 12.5%, 12.9 and 11.5% for granulocytic cells.
Cell lineage involvement in AML patients:
The outcome of FISH studies on erythroid cells, on granulocytic cells and on blast cells in five patients with a cryptic anomaly are shown in Table 2. The majority of blast cells (76–94%) carried the chromosome anomaly in four patients investigated by this method, whereas in one case (No. 1) 46% of the blasts were affected (Figure 1). The granulocytic lineage was found to be involved by each individual chromosome anomaly in all patients, 17–30% of the granulocyte precursors (ie promyelocytes, myelocytes and metamyelocytes) showing one fluorescent signal when hybridized with the relevant probe. In contrast, neutrophils showed a normal hybridization pattern in all patients but one (No. 8), who had 20% of granulocytes with +8.
Erythroid involvement was detected in a minority of cells in three patients, whereas in the remaining two cases the percentage of erythroblasts with an abnormal hybridization pattern was below the sensitivity limit of this technique.
Clinical features of patients with cryptic abnormalities
Clinical and hematologic features in eight patients with cryptic anomalies are presented in Table 3. Five patients had been exposed in the workplace to organic solvents (two cases), pesticides (two cases) and petroleum products (one case), in the absence of effective protection measures. The cumulative life-time exposure index, calculated according to the formula h/day × days/year × years, was >2400 h.23 Only 3/19 patients without a cryptic chromosome anomaly were found to have been anamnestically exposed to myelotoxic agents (P = 0.026).
The percentage of BM blasts was lower in patients with chromosome anomalies than in those without cryptic aberrations (range 36–78%, median 51% vs 40–99%, median 80%). Patients less than 80 years of age having no major contraindication to cytotoxic therapy were treated by an anthracycline drug, or mitoxantrone, plus cytarabine in conventional doses, with or without etoposide, according to the guidelines in use at our institution during the study period. None of the six patients with a cryptic anomaly achieved CR, as compared with 7/12 similarly treated patients without anomalies at the analyzed chromosome loci (P = 0.024).
Forty to 50% of patients with AML (AML-M3 excluded) show an apparently normal karyotype upon CCA, and the identification of cryptic genetic lesions in such cases may have important clinicobiological implications.26,27
Unbalanced chromosome rearrangements leading to gain or loss of chromosome material account for 60–70% of cytogenetically abnormal cases in AML.28,29,30 In approximately half of these cases a chromosome deletion, or a trisomy, may define the stem line, and these aberrations are known to impact considerably on response to therapies and prognostic outcome.29
Occasionally, a few cytogenetically normal AML patients were found to carry an unbalanced chromosome aberration when investigated by molecular cytogenetic techniques.20,31 However, no systematic study has analyzed the incidence and significance of this phenomenon.
In this analysis we included patients with different types of AML (ie those occurring de novo in the adult population, and those occurring in the elderly or preceded by an antecedent MDS), showing upon CCA a normal karyotype in more than 20 cells when analyzed by experienced personnel at referral laboratories. Moreover, all cases in the present series were also found to lack major gene rearrangements by RT-PCR and Southern blot analysis.. The incidence of normal karyotype in the current GIMEMA trial is 46.5% in more than 300 cases successfully analyzed, a figure in line with previous studies of AML excluding APL.28,29,32 The inclusion in the GIMEMA study of routine molecular diagnostics to unravel major fusion genes did not significantly increase the fraction of AML cases with genetic alterations (unpublished observations).
The panel of probes selected for this interphase FISH analysis included the most frequent numerical aberrations and deletions in AML. We estimated that 60–70% of all unbalanced rearrangements found in the analysis of the MRC group would be detected by this approach.29
We did not find anomalies at the chromosome loci analyzed by interphase FISH in 55 adult patients with de novo AML. In contrast, 8/27 (29.6%) elderly AML or AML/MDS were found to carry an occult deletion (seven cases) or an aneuploid clone (one case) (P < 0.0001). This phenomenon was apparently more frequent in the AML/MDS group (five out of six cases with occult anomaly) than in elderly AML (three out of 21 with anomaly), but we were not able to perform a statistical analysis to prove this point due to the small sample size.
Thus, the occurrence of cryptic chromosome deletions in the order of a few hundred kb magnitude, affecting chromosome bands 5q31, 7q31, 17p13 and +8, may be a relatively frequent phenomenon in MDS-related AML and in elderly AML, whereas it appears to be very rare in adult de novo AML. As previously reported in MDS,16 CCA proved unable to detect these anomalies because the size of the deleted segment was beyond the resolution power of banding analysis in the majority of cases. In two cases (Nos 6, 8), however, a low in vitro mitotic index of the abnormal clone was the most likely explanation accounting for the discrepancy between CCA and FISH results.
The occurrence of occult chromosome anomalies in AML was studied by Andreasson and colleagues,20 who used probes for the detection of 12p12 lesions in 19 de novo AML with normal cytogenetics and found two cases – one infant leukemia and one elderly patient – to carry a deletion. In a more recent study using 24-color spectral karyotyping one case carrying a minor clone with −7 was found in 28 AML over 55 years of age.31 These data support our findings and suggest that leukemogenesis in adult de novo AML with normal karyotype may involve cytogenetic alterations not detectable by this FISH approach.26,27 By contrast, our data on post-MDS AML and its biologically related form, elderly AML,21,33,34 show that DNA loss may involve chromosomes 5q/7q and, less frequently, 17p in MDS evolving into AML despite an apparently normal karyotype. FISH may also reveal aneuploid clones (+8) missed by CCA due to technical failure. This finding shows that pathogenetic mechanisms entailing loss of chromosome material may be operative in a fraction of MDS-related AML carrying an apparently normal karyotype. The data by Xie and colleagues35 and by Mori and colleagues,36 who used loss of heterozygosity (LOH) studies, support this argument, showing that DNA segments, located on chromosomes 1p36, 6q, 7p, 10p, 11q, 14q, may be deleted during the evolution of preleukemia into leukemia. Recently, LOH at 5q22.3-35.3, 11p15 and 19q12 loci was found in four out of 18 childhood AML cases with normal karyotype.37
Overall, these findings are reassuring with respect to the sensitivity of CCA in detecting unbalanced chromosome rearrangements in adult de novo AML, while they suggest that interphase FISH may have a role in detecting chromosome rearrangements in elderly AML and MDS/AML with an apparently normal karyotype. It is worth noting that the presence of cryptic chromosome rearrangements was found to have important clinicobiological implications in a recent study including unselected cases of de novo MDS.16 Our patients with cryptic anomalies were anamnestically exposed to myelotoxic agents in the workplace more frequently than the remaining patients in the elderly AML and MDS/AML group. This finding is consistent with previous observations of an existing association between environmental exposure and development of unbalanced chromosome changes.38,39,40,41 As expected, our patients with chromosome deletion and trisomy proved to be less responsive to induction therapy than other patients in the same group.42 These considerations suggest that cryptic 5q and 7q deletions may show similar clinicobiological correlations as compared with gross cytogenetic rearrangements involving these chromosome segments.
Because the proportion of FISH abnormal cells in this study was in the 33–60% range, we adopted a direct hybridization procedure preserving cell morphology25 to clarify the pattern of cell-lineage involvement by each individual chromosome lesion. Although heterogeneity of lineage involvement by specific chromosome aberrations was documented in myeloid neoplasias,15,43 our data show a relatively lineage-specific distribution of cryptic chromosome lesions. Indeed, with the exception of patient No. 8, in whom a sizeable fraction of erythroblasts were shown to carry trisomy 8, all our patients showed a preferential distribution of the chromosome anomalies within the blast cell population. The blast cells seemingly maintained limited maturation capability along the granulocytic lineage, with a minority of promyelocytes–myelocytes carrying the chromosome anomaly. Neutrophils were not affected by the chromosome anomaly. These findings suggest that the acquisition of each individual chromosome lesion was associated with clonal evolution resulting in the progressive accumulation of cytogenetically abnormal blast cells with consequent transformation of MDS into AML.
In conclusion, we have shown that: (1) occult chromosome deletions affecting chromosome 5q, 7q and 17p, and trisomy 8, may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; and (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.
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This work was supported by AIRC and AIL, by MURST 1999 ex 40% and 60%, by CNR. The authors thank the following physicians for referring BM samples for cytogenetic analysis: S Amadori (Rome), G Avanzi (Novara), P Bodini (Cremona), AM Carella (S Giovanni Rotondo), Epis (Sondrio), G Fioritoni (Pescara), A Gabbas (Nuoro), A Levis (Alessandria), M Longinotti (Sassari), R Mozzana (Gallarate), F Nobile (Reggio Calabria), A Peta (Catanzaro), M Petrini (Pisa), B Rotoli (Napoli), G Torelli (Modena), E Volpe (Avelllino).
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