Biphenotypic acute leukemia (BAL) is a rare, difficult to diagnose entity. Its identification is important for risk stratification in acute leukemia (AL). The scoring proposal of the European Group for the Classification of Acute Leukemia (EGIL) is useful for this purpose, but its performance against objective benchmarks is unclear. Using the EGIL system, we identified 23 (3.4%) BAL from among 676 newly diagnosed AL patients. Mixed, small and large blast cells predominated, with FAB M2 and L1 constituting the majority. All patients were positive for myeloid (M) markers and either B cell (B) (17 or 74%) or T cell (T) (8 or 34%) markers with two exceptional patients demonstrating trilineage phenotype. Six (50%) of studied M-B cases were positive for both IGH and TCR. In six (26%) patients myeloid lineage commitment was also demonstrable by electron cytochemistry. Abnormal findings were present in 19 (83%) patients by cytogenetics/FISH/molecular analysis as follows: t(9;22) (17%); MLL gene rearrangement (26%); deletion(6q) (13%); 12p11.2 (9%); numerical abnormalities (13%), and three (13%) new, previously unreported translocations t(X;6)(p22.3;q21); t(2;6)(q37;p21.3); and t(8;14)(p21;q32). In conclusion, the EGIL criteria for BAL appear robust when compared against molecular techniques that, if applied routinely, could aid in detecting BAL and help in risk stratification.
Human acute leukemias (ALs) are broadly classified as myeloid or lymphoid according to the expression of surface and/or cytoplasmic antigens. Uncommonly, the lineage of origin is not clear; either two separate blast populations are encountered, one myeloid and the other lymphoid, or, a single blast population demonstrating evidence of both myeloid and lymphoid differentiation concurrently. The new WHO classification system refers to the former entity as bilineal AL and to the latter as biphenotypic AL (BAL), and categorizes them as subtypes of the group ‘AL of ambiguous lineage’.1, 2 Knowledge about BAL is limited, both in terms of clinical and biological presentation and also with regard to treatment outcome. It is generally believed that BAL is derived from marrow stem cells with the capacity for expressing antigens from more than one cell line.3, 4 However, there are, as yet, few specific studies delineating the fundamental molecular characteristics of BAL. The prevalence of BAL has ranged from 2% to almost 20% of ALs in various reports.5, 6, 7 This wide variability may be attributed to a number of reasons including a lack of consistent diagnostic criteria, use of differing panels of antibodies, and failure to recognize the lack of antigen specificity of some of the antibodies used.
Definition of BAL using objective criteria is important in order to distinguish this unusual type from ALL or AML with aberrant expression of markers of other lineages. The European Group for Immunological Classification of Acute Leukemia (EGIL) has proposed a scoring system for assigning cell lineage8 in AL. This system is based on the number and degree of specificity of myeloid/lymphoid antigens expressed by the leukemic blasts. Accordingly, BAL is diagnosed when scores for both myeloid and lymphoid lineages are greater than two points; rare cases may show tri-lineage differentiation.
Myeloid and lymphoid markers that are expressed in lymphoid and myeloid leukemias, respectively, but do not reach greater than two points are referred to as aberrant expression. Few studies have analyzed the diagnostic and prognostic factors, clinical and biological presentation of BAL patients when compared to other ALs.5, 6, 9, 10, 11
Herein we have utilized morphological, immunophenotypic, cytogenetic and molecular genetic characteristics to assess whether the EGIL scoring system is sufficiently robust, in practice, to define BAL.
Materials and methods
Materials from 676 consecutive patients with AL presenting at our hospital between January 2000 and May 2005 were evaluated. From reviewing the immunophenotypic database, 23 cases (3.4%), classified according to the EGIL scoring system as BAL, were identified. The diagnosis was based on initial morphological and cytochemical evaluation that was then supported by extensive immunophenotyping.
Wright–Geimsa-stained peripheral blood and bone marrow aspirate smears from all patients were reviewed, together with a slide from each bone marrow stained for either myeloperoxidase (MPO) or Sudan black B (SBB) and hematoxylin–eosin core bone marrow biopsies. The materials were examined by a consultant hematopathologist.
Immunophenotyping was determined on mononuclear cells isolated immediately after collection of bone marrow aspirate or peripheral blood samples. Briefly, an aliquot from the aspirate/peripheral blood was washed twice using PBS buffer with 1% BSA and 0.1% NaN3. Subsequently, the aspirate was filtered and the count adjusted to 0.5–1 × 106. Fifty 50 μl aliquots were then placed in tubes containing appropriate combinations of monoclonal antibodies and incubated for 15–20 min in the dark at room temperature. Subsequently, the red cells were lysed using Coulter Lysing Kit (Immunolyse) followed by a single wash in PBS buffer. The cells were re-suspended in 0.5 ml of 1% paraformaldehyde and stored in the refrigerator prior to analysis. For staining of cytoplasmic and nuclear antigens, the IntraPrep Permeabilization Reagent kit (Beckman Coulter Inc., Fullerton, CA, USA) was used, strictly following the recommendations of the manufacturer. The cells were analyzed on a FACS Caliber flow cytometer (Becton Dickinson Immunocytometry systems, San Jose, CA, USA). Data acquisition was performed using Cell Quest software (Becton Dickinson) where minimums of 10 000 events per tube were acquired. Instruments were calibrated daily using CALIBRITE beads (Becton Dickinson) and two levels (normal and low) of controls using CD3/CD8/CD45/CD4, and CD3/CD16+CD56/CD45/CD19. The blast cells were gated using CD45 vs SSC technique, excluding non-viable cells as well as intact mature nucleated cells. A sample was considered positive for surface antigens if more than 20% of the leukemic cells expressed fluorescence intensity more than 98% of the negative control cells. Positivity for TdT and cytoplasmic antigens was arbitrarily defined as more than 10% of the cells exhibiting nuclear or intracytoplasmic fluorescence as compared with negative controls. The criteria for diagnosis of BAL were based on the EGIL scoring system.8
Monoclonal antibody panels
T-cell markers: CD1a-PE (Coulter), CD2-PE (BD), CD3-FITC (BD), CD4-APC (BD), CD8-PE (BD), CD7-FITC (BD), CD5-FITC (BD), TCRαβ-FITC (BD), TCRγδ-PE (BD); B-cell markers: CD19-APC (BD), CD20-PE (BD), CD22-APC (BD), Kappa-FITC (BD), Lambda-PE (BD), IgG-FITC (Biosource International Camarillo, CA, USA), Intracytoplasmic μ chain – FITC (Biosource); The myeloid markers used were: CD14-FITC (BD), CD13-PE (BD), CD33-PE (BD), CD64-FITC (BD), CD56-FITC (BD), CD117-PE (BD), CD15-FITC (BD), CD65-FITC (Coulter) and CD11c-PE (BD); Monoclonal antibodies against cytoplasmic and nuclear antigens used were: MPO-FITC (Coulter), CD79a-PE (Coulter), CD3-FITC (Coulter). TdT-FITC ‘polyclonal’ (Coulter) was used to detect nuclear TdT; Additional mAbs panels used were: CD45-PerCP (BD), HLA-DR-FITC (BD), CD34-APC (BD), CD10-FITC (Coulter), CD11b-PE (BD), CD9-FITC (BD), CD56-APC (BD), CD61-FITC (BD), and CD71-FITC (BD).
Ultrastructural MPO (EM-MPO)
Leukocytes, isolated in vitro from blood or bone marrow using the ficoll-paque gradient centrifugation method, were fixed in 1.6% gluteraldehyde and incubated with benzidine dihydrochloride to form complexes with MPO present in the granules of early myeloid cells. Cell pellets were embedded in resin, and thin sections cut at 0.7 μm were mounted on copper grids. Blast cells were viewed unstained, under a Phillips 301 electron microscope.
Chromosome analysis from bone marrow aspirate cells was performed according to our standard laboratory techniques. Briefly, 18 and/or 24 h cultures were set up in RPMI 1640 culture medium (cat.# 59-202-77, JRH Biosciences, USA), supplemented with 20% fetal bovine serum (cat.# 12-106-77P, JRH Biosciences, USA), 12% GCT conditioned medium (Cat.# 50-0815, IGEN, USA), 2% L-glutamine (200 mM, Cat.# 59-202-77, JRH Biosciences, USA), 5000 μg/ml gentamicin (Cat.# G1522, Sigma, USA). Cultures were incubated at 37°C in 5% CO2 environment and harvested after a short treatment with ethidium bromide (1 h, 5 μg/ml), colcemid (1 h, 10 ng/ml, Cat.# 9311, Irvine Scientific, USA), cancer hypotonic solution (1 h) and a 3:1 methanol acetic acid mixture. At least 20 GTG-banded metaphases were used for karyotype analysis. Definition of a clone and karyotype designation was according to ISCN (1995).
Red cells of blood and bone marrow specimens, collected in EDTA, were lysed and the nucleated cell pellet transferred to DNAZOL and TRIZOL. Proteins were digested by a guanidinum-based solution and nucleic acid separated by selective precipitation using isopropanol. Isolated nucleic acid was evaluated for quality and quantity using agarose gel electrophoresis as well as spectrophotometer readings at 260 and 280 nm. To determine the clonality of B cells, immunoglobulin heavy chain (IgH) gene rearrangement polymerase chain reaction (PCR) assay, using a single primer set for the germline variable (VH) and joining (JH) pair was used. To determine the clonality of T cells, PCR assay using three sets of primers, Vγ(1–8), Vγ(9) and Vγ(10–12) from the T-cell receptor (TCR) gene locus were used. For the detection of BCR/ABL P210 mRNA, total RNA was reverse transcribed. The generated cDNA was amplified with forward primer located in the BCR exon b2 to combine with the reverse primer in the ABL exon a4. The fluorescent hybridization probe pair used is located in the ABL exon a3. The location of primers and hybridization probes also allowed the amplification and detection of the b2a3 and b3a3 fusion transcripts. The amount of fluorescence resulting from the two probes is proportional to the amount of PCR product. For BCR/ABL P190, a nested PCR was used to amplify BCR-ABL cDNA after reverse transcription of mRNA (see Table 1).
For FISH analysis, LSI bcr/abl ES, Cat.# 32-191022; LSI MLL gene dual color probe, Cat.# 32-190083 and X and Y probes Cat.# (Abbot-Vysis Inc., Downers Grove, IL, USA) were used to detect BCR/ABL, MLL rearrangement and loss of sex chromosomes in the interphase cells. Pre- and post-hybridization procedure was performed according to the manufacturer's recommendations (Abbot-Vysis Inc., Cat. 33-270000). Interphase cells were viewed for the target signals using a Zeiss Axioplan 2 fluorescence microscope equipped with; (a) 103w/2 HBO mercury lamp and DAPI/Green/Orange triple band pass filter and (b) cytovision image analysis/Z-stack focus motor software system (Applied Imaging, UK). At least 100–200 cells were analyzed for target signals.
Collected data were entered into the statistical software SAS (SAS Institute Inc., version 9). The mean, median, range, frequency and standard deviation (s.d.) and percentage were used as descriptive statistics.
From 676 cases of both adult and pediatric patients of AL diagnosed and treated in our institute over a period of more than 4 years, 23 cases (3.4%) were diagnosed as BAL according to the EGIL criteria.8 Fourteen were males and nine were females with male to female ratio of 1.5:1. Eleven patients were 14 years or less (the cutoff age for pediatric patients in our institute) and the other 12 cases were classified as adults.
The mean WBC count on presentation was 56.03±54.2 × 109/l, hemoglobin 90.9±18.8 g/l, platelet 80.4±55.36 × 109/l, blasts ±58.22%±31.1 and LDH 1649.2±1287.8 μ/l. Six patients had laboratory features of disseminated intravascular coagulopathy. Thirteen out of 23 BAL cases (56.5%), showed mixed morphology with a spectrum of small and large size blast cells. Ten of these were granular and in one case Auer rods were present. The rest of the cases were either small blasts (six cases) or large blasts. Based on FAB classification the most frequent type was M2 followed by L2 (Table 2).
A panel of 25 monoclonal antibodies was used to phenotype each case. Fifteen out of 23 cases (65%) had myeloid and B-lymphoid phenotype, six cases (26%) had myeloid and T-lymphoid phenotype and two cases (9%) had trilineage phenotype. All cases had an EGIL score of >2 in each lineage (Table 3). For myeloid lineage, the most frequently observed positive markers were: CD33 in 19 patients (82%), CD13 in 18 patients (78%), CD15 and CD117 in 14 patients (61%). For B-lymphoid phenotype, the frequently encountered markers were: CD19, CD79a and CD22 in 13 patients (76%) and CD10 in nine patients (53%). The most frequently found positive T-lymphoid markers were: cyCD3 in eight (100%) cases, CD7 in seven (88%) cases and CD2 in six (75%) cases. CD45 and stem cell marker CD34 were positive in 22 (96%) patients while TdT was positive in 18 (78%) patients. CD11b was positive in seven cases (30%).
MPO activity was evaluated by flowcytometry and/or cytochemistry in all cases and in nine cases by EM-MPO. MPO was positive in seven patients by flowcytometry and in six by cytochemistry (MPO or SBB) while in four others it was positive only by EM-MPO. Only in one case was there agreement between all three methods. Detailed description of the markers with comparison with other similar studies is given in Tables 3 and 4.
Cytogenetic analysis was successful in 22 (96%) out of 23 cases. Fifteen (68%) patients showed a clonal abnormality and seven (32%) had normal karyotypes (Table 5). Details of karyotypes are given in Table 2. Philadelphia chromosome t(9;22) was present in two patients; both these cases expressed B-lymphoid and myeloid antigens. Structural rearrangement of (1) 11q23 was found in three cases co-expressing myeloid and B-lymphoid antigens; (2) deletion (del)(6q) in three cases, two with myeloid/T lymphoid and one with myeloid/B lymphoid and (3) 12p11.2 in two cases. Numerical abnormalities were seen in three cases: trisomy 4 (case #22), trisomy 19 and X (case #2) and loss of Y chromosome (case #1). Complex karyotype with ploidy greater than 2n or 3n was observed in one case. In three cases, translocations not previously reported in AL were found and were t(X;6)(p22.3;q21) in case #3, t(8;14)(p21;q32) in case #17, and t(2;6)(q37;p21.3) in case #16, respectively (Table 5).
Table 3 shows the results of molecular analysis in BAL patients. IgH and TCR rearrangement was observed in six out of 12 (50%) cases of myeloid/B-lymphoid BAL and TCR was rearranged in two out of five (40%) cases of myeloid/T-lymphoid BAL. Four cases (33%) out of 12 were positive for P190 and none for P210.
By FISH analysis, MLL gene rearrangement was found to be positive in six (50%) out of 12 cases analyzed (three of these cases also showed 11q23 abnormality by cytogenetics) and BCR/ABL rearrangement was present in two out of 13 cases (13%), both of these cases were also positive for t(9;22) by cytogenetic analysis.
Although BAL is a rare entity, it has recently gained some significance especially with the availability of objective diagnostic criteria. Earlier reports of BAL showed variability in the prevalence,6, 12, 13 however, recently, mainly due to objective diagnostic criteria, the prevalence of BAL has shown some consistency (2–5%).5, 14, 15 In our study, the prevalence of BAL is 3.4%, which is in agreement with previous published reviews of BAL.5, 14, 15 Although the morphological analysis of AL now has less emphasis, it continues to be the initial screening procedure that could raise possibility of BAL, by demonstrating the presence of a mixed population of cells.
The co-expression of myeloid with B-lymphoid antigens in the present study represents 65% of all BAL while the co-presence of myeloid and T lymphoid represents only 26% and the trilineage phenotype is a rare event.
Our data are similar to previously reported series9, 14, 15 (Table 4). The theory that the cell of origin in BAL is an early hematopoeitic stem cell, is supported by nearly all cases of BAL in our study expressing stem cell marker CD34. Although myeloid markers, CD13 and CD33 are less specific (each equivalent to a score of 1 by EGIL) yet they are more frequently encountered in BAL patients and their inclusion in the analysis of cases of AL would result in better detection rate of BAL. We also demonstrate here the superior sensitivity of EM-MPO over both flow cytometry and cytochemistry in detecting MPO activity as was similarly demonstrated by Shetty et al.16 Thus, we would highly recommend the inclusion of EM-MPO as a supplementary criterion, when available, for inclusion with the other EGIL criteria for AL classification.
We report positivity of CD11b in seven cases (30%) suggesting its potential use as a myeloid marker, however, further studies are recommended to evaluate its use for this purpose. The frequency of TdT positivity in our study is 78%, comparable to previous reports (Table 4).
Cytogenetics of myeloid and lymphoid leukemias has established specific recurrent chromosome abnormalities, which are utilized for both diagnostic and prognostic purposes. However, very few cytogenetic studies are available in BAL9, 11, 15, 17 (Table 5). A review of these studies along with our data shows a high incidence of clonal chromosomal abnormalities. Among these, t(9;22) appears to be high (28–35%),9, 10, 14 however, the incidence in our study was lower (9%). The use of molecular studies resulted in better detection of the abnormality (17%). The incidence of MLL gene rearrangement was also reported to be higher (10–32%).9, 10, 14 Although our data showed a lower incidence (13%), however, by combining FISH analysis, the incidence is comparable to published reports (26%). Del(6q) abnormality is known to be commonly associated (10–20%) with childhood T-cell ALL and in adult ALL; the frequency is comparatively lower (5%) and is reported predominantly in young adults (aged 15–40 years).18 In our series of BAL, del(6q) was present in 13% patients; all of these were young adults (16–21 years) consistent with previous reports. Del(12p) was also found to be common in BAL patients in all reported studies. Again, it is important to document 12p abnormality in BAL by molecular techniques to determine its prevalence. Del(5q or 7q as a solitary abnormality is generally associated with myeloid disease but in both the present and previous studies BAL show 5q and 7q abnormalities along with complex karyotypes. As a result, their significance is difficult to explain. We recommend utilizing FISH technique in detecting masked chromosome abnormities of prognostic importance in BAL with apparently normal karyotype.
Previously we have reported trisomy 4 in a case of acute BAL with T-lineage markers.19 In the present study, another case of trisomy 4 with a secondary abnormality (14q32) was found with co-expression of myeloid and B- and T-lymphoid antigens. The difference in the immunophenotypes of the present case with our previously reported patient could be due to the presence of secondary abnormality – add(14q32). Loss of sex chromosome (Y) was seen in one case in our study and also in two cases in the series of BAL reported by Killick et al.14 The significance of this abnormality in BAL needs to be elucidated with similar additional cases. Complex karyotypes with ploidy >2n or 3n were observed in 1–2 cases in all the studies including the present one. These cases presented with secondary leukemia.
Occurrence of new translocations has also been reported in 14% of BAL patients.10, 14 In the present series three cases (13%) revealed new translocations prevalence similar to previous reports. It remains to be seen if any specific gene/genes in these new translocations are of special significance in BAL.
IGH and/or TCR gene rearrangements were reported previously in few cases of BAL. Buccheri et al.13 had reported positivity for both in four out of nine cases of BAL Interestingly, IgH rearrangements are reported in 70–90% of B-cell precursor acute lymphoblastic cases (BCP-ALL) and TCR gamma had been described in 40–70% of BCP-ALL.20, 21 Failure in amplifying IgH due to various reasons had been reported in up to 20% of cases of BCP-ALL.22 Furthermore, Brumpt et al.20 and Szczepanski et al.23 demonstrated the significance of rearrangement of both IgH and TCR as valuable markers for follow-up in BCP-ALL, yet the significance of molecular findings in BAL have not been studied.
In summary, we present a relatively large cohort of patients with BAL, representing 3.4% of our cases of AL. We demonstrate that the EGIL system for assigning BAL is very robust, since it was supported by consistent molecular findings in 75% of cases. We also show that EM cytochemistry could be a useful adjunct in detecting myeloid lineage when other methods are unsuccessful. It might therefore be worthwhile to include this modality in evaluating patients with suspected BAL when this modality is available.
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Owaidah, T., Beihany, A., Iqbal, M. et al. Cytogenetics, molecular and ultrastructural characteristics of biphenotypic acute leukemia identified by the EGIL scoring system. Leukemia 20, 620–626 (2006). https://doi.org/10.1038/sj.leu.2404128
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