Clinical immunology

Rare types of acute leukaemia - analysis of immunophenotype of leukaemia cells

Introduction



The acute leukaemia is heterogeneous group of lymphoproliferation of haematopoietic bone marrow cells with clonal characteristic. Based on immunophenotype of leukaemia cells the two main types of leukaemia are described: acute lymphoblastic leukaemia T or B cell lineage (ALL-T, ALL-B) and acute myeloblastic leukaemia (AML). The leukaemia cells of ALL are from B cell or T cell lines and their immunophenotype resemble the maturation and differentiation stages of normal ontogenesis of T or B lymphocyte. The immunophenotype of leukaemia cells identical to normal haematopoiesis stages is described as typical. The all kinds of disturbances in surface expression of determinants are included into a group of atypical immunophenotype. Till last decade the typical immunophenotype in acute leukaemia were noted in more than 80% of cases. Now, improved methods like immunophenotyping with flow cytometry and genetic studies including fluorescent in situ hybridisation show higher percentage of atypical immunophenotype of leukaemia cells. However, the precise study of normal haematopoiesis showed expression of determinants admitted to be restricted to one line on normal cells on the another line (lymphoid on myeloid and vice versa) [1-5]. This type of specific co-expression on leukaemia cells is now concerned rather as proliferation of cells in this exact stage of haematopoiesis than atypical immunophenotype. The true atypical immunophenotype includes the following disorders:

- lack of expression of determinant characteristic for given stage of haematopoiesis (incomplete immunophenotype),

- simultaneous expression of determinant from precursor and mature stage of development (asynchrony of immunophenotype),

- hyperexpression of determinant,

- co-expression of one or two determinants from other line than origin of leukaemia cells (e.g. CD19 on AML, CD33 on common ALL, CD13 on ALL-T) [3-10].
These forms of atypical immunophenotype are based on surface expression of determinants on leukaemia cells with precisely described origin. The another group of leukaemia is associated with disturbed development of stem and lineage committed cells. These types of leukaemia are difficult for diagnosis and classification based on immunophenotype. This group includes following rare forms:

- bi-phenotypic acute leukaemia (BAL) when the one population of cells shows balanced expression of myeloid and lymphoid determinants (noted as i.e. AML/ALL-T),

- bi-lineage leukaemia when the population of leukaemia cells is heterogeneous morphologically with expression of determinants from two lines on respective subpopulations,

- bi-clonal leukaemia - concurrent presence of two clones of leukaemia cells with a different immunophenotypes (noted as AML + ALL-T),

- acute mixed lineage leukaemia (AMLL) - the balanced expression of determinants from different lines. The morphology of these cells is more likely early myeloid or mixture of myeloid and lymphoid [2-4, 7-13]. The classification is often very difficult.

The classification of bi-phenotypic leukaemia and bi-lineage leukaemia is often difficult as these names are used without differentiation. The precise classification requires strong co-operation between persons responsible for morphological and immunophenotypic classification.
These rare forms are more frequent in children with immunodeficiency (Wiskott-Aldrich, ataxia-teleangiectasia) or Down syndrome [9, 10].

This study shows the flow cytometry analysis of immunophenotypes of leukaemia cells in rare forms of acute leukaemia at diagnosis. In some cases the immunophenotypes during course of disease are included.



Material and methods



The bone marrow or peripheral blood specimens derived from 10 children admitted in the Department of Oncology and Haematology and 2 adults from the Department of Haematology admitted with initial diagnosis of acute leukaemia were subjected to routine diagnostic procedure. The bone marrow was taken under anaesthesia from iliac crest and immediately solved in medium with EDTA (0.1 ml of 10% EDTA in 2 ml BSS - Gibco, Paisley, Scotland). The peripheral blood (1-3 ml on 0.1 ml of 10% EDTA) was included in cases with high leukocytosis consisting more than 90% of leukaemia cells.



Staining with monoclonal antibodies



The leukaemia cells were isolated from bone marrow or peripheral blood with density gradient on Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) 400 g, 20 min, room temperature). The cells from interphase were collected, washed in buffered saline (PBS, Sigma-Aldrich, Germany) and counted with trypan blue exclusion dye. The cells were suspended in PBS at concentration 2x106/ml and distributed into tubes (50 ml/tube) with monoclonal antibodies and isotypic control FITC or RPE labelled (Dako, Glostrup, Denmark) on the bottom.

The following combination of monoclonal antibodies was used:

- mouse immunoglobulin IgG11 FITC/IgG2 RPE (isotype control),

- anti-CD2 FITC/CD19 RPE (T or B cell lineage),

- anti-HLA-DR FITC/ CD3 RPE (precursor cell or mature T cell),

- anti-CD4 FITC/CD8 RPE (T cell lineage or myeloid - CD4),

- anti-CD7 FITC/CD5 RPE (T cell lineage, early myeloid - CD7),

- anti-CD10 FITC/CD22 RPE (precursor or mature B cell),

- anti-CD 13 FITC/CD33 RPE (myeloid cell),

- anti-CD34 FITC or perCP (precursor cell),

- anti-sIg (GAM) FITC (surface immunoglobulins - mature B cell),

- anti light chain kappa FITC/ lambda RPE (mature B cell).

This combination allowed us to assay the expression of two determinants on cell surface. This combination was changed to prove the unusual co-expression observed in screening assay of individual patient. The final description of immunophenotype of leukaemia cells was based on two-steps procedure - screening test with routine set of monoclonal antibodies and double staining for unusual expression of determinants to prove the classification.
The cells were incubated 30 min, at 4oC, in dark with monoclonal antibodies and washed twice thereafter. The pellet of cells was suspended in 0.4 ml PBS and assayed in flow cytometer (FACScan V and FACS Calibur, Becton-Dickinson, Mountain View, CA).
Cytoplasm staining was performed in selected cases when the surface expression did not allow determining the origin of leukaemia cells. It included staining for myeloperoxidase (MPO - myeloid origin), CD22 or CD79a (B cell origin) and CD3 (T cell origin). The washed cells from interphase were fixed with Cytofix/Cytoperm (Becton-Dickinson) and stained as for expression of determinants in cytoplasm. The isotype control was run in parallel. After incubation the cells were washed and assayed in flow cytometer.



Analysis in flow cytometer



The routine analysis included:

- description of cell population based on FSC and SSC characteristics,

- percentage of given cell population within whole cell suspension,

- assay of immunophenotype of selected (gated) population of leukaemia cells,

- assay of fluorescence intensity of leukaemia cell population as compare to normal bone marrow counterparts. It is in relationship to expression of determinant on the surface.

Ten thousand events were collected form each sample without life gating with stable compensation and other parameters of cytometer. The dot-plot with FSC and SSC was used for analysis of present populations of cells, for gating of leukaemia cell population, gating of populations with special interest e.g. unusual immunophenotype. The population of leukaemia cells was considered when the cells with atypical morphology were observed and/or atypical, unique population replaced the normal cells. In ALL the population of leukaemia cells usually consisted of 90-98% of bone marrow cells, in AML - 40-80% of bone marrow cells. The expression of given determinant was consider when intensity of fluorescence of at least 20% of cells was higher than isotype control. The single expression of determinant was analysed with histogram statistics; the double expression was analysed with quadrant statistics. The comparison of fluorescence intensity was performed with overlay of histograms. The expression of combination of determinants was analysed on populations of leukaemia cells gated based on expression of given determinants (i.e. co-expression of CD33 was analysed on CD19+ cells).



Results



The study included 10 children (2.5–15 year of age) and 2 adults with initial diagnosis of acute leukaemia. The immunophenotypes of leukaemia cells are shown in Table 1. Some clinical data of patients included into this study are presented in Table 2. The first three cases (patient no. 1-3) represented true acuted mixed lineage leukaemia (AMLL), the following 4 cases (patient no. 4, 6-7) represented co-expression of determinants from other cell lines and the remaining cases were classified as bi-phenotype (patients no. 5, 11-12) and bi-lineage leukaemia (patients no. 8 and 10). The patient no. 9 represented very rare case of co-existence of NHL and AML.

Patient no. 1 was a boy admitted at the age of 6 with the hepatosplenomegaly and hyperleukocytosis. The immunophenotype of leukaemia cells showed expression of T-cell lineage determinants (CD4, CD7), B cell lineage (CD22) and myeloid (CD33, CD13, CD15) and early precursors (CD34). The cytoplasm expression of CD3 suggested T line origin of leukaemia cells. The morphology of leukaemia cells was close to lymphoid progenitors. The classification of this case as AMLL was based on high expression of CD22 in addition to expression of myeloid determinants on T cell origin leukaemia cell. The complete remission was noted after 17 weeks of the induction therapy according New York’97 protocol [14] followed with different combinations of cytostatics because of resistance to therapy of leukaemia cells. The resistance to therapy supported the diagnosis of AMLL. The first remission lasted 6 months followed with mixed relapse (CNS, testes and bone marrow). The second remission was obtained after 4 weeks of intensive chemotherapy. The next relapse was after 3 months with following short-time remission. In third remission the mobilisation of PBSCT was performed. After autoBMT the next relapse occurred within one month. Patient died due to leukaemia progression. The immunophenotype of leukaemia cells during first relapse showed expression of CD33, CD15, CD4 (myeloid population) and CD10, CD19, CD22, HLA-DR (lymphoid population) without CD7, CD34 observed at the beginning. The leukaemia cells within (central nervous system fluid) CSF showed expression of CD33, CD34 and CD22 (Fig. 1). During the last relapse (after autoBMT) the leukaemia cells showed expression of CD33, CD15, CD7, HLA-DR without CD22. The expression of myeloid determinants was stable during whole course of the disease. The expression of multidrug resistance molecule (MDR) was noted in the acute phase of disease. Expression of determinants from B cell and T cell lineage was changing (Table 3). It suggests the different chemosensitivity or chemoresistance of subpopulations of leukaemia cells or the defect in stem cells what resulted in different immunophenotype during course of the disease. Unfortunately, we have no genetic studies of leukaemia cells in this patient.

The leukaemia cells of patient no. 2 (AMLL) showed an expression of determinants on part of morphologically declared leukaemia cells. There was one population of cells under microscope and on FSC/SSC dot plot. The immunophenotype included expression of determinants from myeloid, B cell and T cell lineage. The expression of CD3 within T cell lineage determinants was noted as rare phenomenon. The patient demonstrated symptoms of ataxia-teleangiectasia. After complete leukaemia therapy he remains in remission with a progression of ataxia-teleangiectasia symptoms (Table 2).

Patient no. 3 was a girl with Down syndrome and AMLL. The immunophenotype of leukaemia cells was unusual and the expression of CD2, CD8 and CD19 and CD22 and CD33, CD13 was observed on the same cells on gated population confirmed by double staining.
The co-expression of determinants from other cell line than line of leukaemia cell origin is most frequent type of atypical immunophenotype. Within our group of patients during last 3 years of observation about 30% of patients demonstrated this type of atypical immunophenotype of leukaemia cells. We decided to show co-expression from more than one line and rare combinations of determinants within immunophenotype of leukaemia cells.
The immunophenotype of leukaemia cells from patient no. 4, 6 and 7 was classified as co-expression of determinants (Table 1). The leukaemia cells of patients no. 4 showed expression of CD13 and CD19 on cell of ALL-T. In double staining the cells CD19+ were within population of CD13+. The expression of CD19 was rare, observed in singular cases, more often expression of CD10 or CD20 was seen. The cells of patient no. 6 expressed CD13 and partially CD2 what was unusual co-expression. After therapy the patients no. 4 and 6 remain in remission (Table 2).
The leukaemia cells from patient no. 5 demonstrated balanced expression of myeloid and T lineage determinants but the morphology was defined as lymphoid. The classification of bi-phenotype leukaemia seemed to be more appropriate than co-expression of determinants. The expression of CD3, CD8 and CD2 suggested T origin of leukaemia cells against only CD13 as myeloid origin determinant (CD4 determinant might belong to myeloid as well as T line origin of leukaemia cell). The origin of cells was determined as myeloid according to expression of MPO and the therapy was for myeloid leukaemia. This case resembled the difficulties in classification of immunophenotype.
Patient no. 7 was admitted with the symptoms of hyperleukocytosis and hepatosplenomegaly. The immunophenotype of leukaemia cells showed expression of CD10, CD19, HLA-DR, CD34 and CD33 and CD13 on leukaemia cells defined as lymphoid by morphology (Table 1 and Table 4). The combination of monoclonal antibodies: CD19 RPE and CD13 FITC showed expression of both determinants on the same cells. After 3 weeks of New York’97 induction therapy the complete remission was reached and lasted 22 months without disease-associated events. In the bone marrow relapse the leukaemia cells showed lymphoid morphology as before but mixed lineage immunophenotype (CD3, CD10, CD19, CD22, CD13, CD33, HLA-DR and CD34). The progression of therapy-resistant leukaemia was the cause of fatal outcome despite of intensive treatment (Table 2).
The patient no. 8 was classified as bi-lineage leukaemia based on expression of MPO and CD3 in cytoplasm of leukaemia cell population assayed with double staining (Table 1). There were no visible subpopulations within leukaemia cell population either on FSC/SSC dot plot or in the morphology. After 3 years of remission the relapse occurred. The immunophenotype of leukaemia cells changed toward to common ALL+My (HLA-DR, CD19, CD10, CD22 and co-expression of CD33). There was no expression of T lymphocyte line determinants and CD34 previously noted. The patient is now under therapy used in relapses of acute lymphoblastic leukaemia.
Patient no. 9 was admitted with hepatosplenomegaly, enlargement of tonsils and right testis, haemorrhage diathesis (typical localisation around eyes). The beginning of the disease was 6 months earlier with the symptoms diagnosed as rheumatoid arthritis treated with steroids and methotrexate. The improvement of symptoms was for short period of time. The enlargement of left testis was diagnosed by histology (without immunophenotype) as non-Hodgkin lymphoma (NHL), probably derived from B cell lineage. On admission the bone marrow consisted of leukaemia cells with morphology of early myeloid and expression of CD33, CD4, CD22, HLA-DR, partially CD15 and MPO (Table 1 and Figure 2). Because of severe clinical symptoms the short treatment was introduced. The discrepancy between earlier diagnosis of NHL-B from left testis and AML from bone marrow indicated the biopsy of right testis. The cells from this biopsy showed expression of HLA-DR, CD33 and CD22 (partially) on the surface and CD22 in the cytoplasm (Figure 3). The clinical diagnose of co-existence of NHL-B (testis) and AML (bone marrow) was determined. The full program of NHL-B therapy was continued and followed with complete remission after 2 weeks. The maintaining therapy was according to ANLL protocol. The bone marrow relapse was noted after following 10 months (Table 5). Nevertheless after intensive second-line chemotherapy, the patient died in clinical symptoms of sepsis.
The immunophenotype of leukaemia cells of patient no. 10 was included (Figure 4) because of mixture of CD79a+ and MPO/CD79a+ leukaemia cells within one population of cells on FSC/SSC dot pot. In the morphology the two populations were clearly visible and the smaller one consisted larger cells with more myeloid characteristic. The flow cytometry analysis did not allow to distinguish these subpopulations of leukaemia cells on FSC/SSC dot plot. The cut-off point for second lineage of leukaemia cells was 10% of cells from other line. In percentage of MPO+ (17.7%) was above this criteria and the bi-lineage leukaemia was determined in this case. After induction therapy this patient remains in complete remission.
The immunophenotype of leukaemia cells of patient no. 11 was a combination of B cell line and myeloid line. The origin of cells was myeloid (Table 1). Despite of expression of multidrug resistance molecule (MDR) the patient reached complete remission after second induction course of chemotherapy followed with auto-PBSCT resulted in complete remission (15 months of observation time) (Table 2).
The immunophenotype of patient no. 12 was similar to previous patient with a good response to therapy (Table 1). The complete remission was obtained after one induction course of chemotherapy. After auto-PBSCT the relapse in CNS was observed and intrathecal chemotherapy and radiotherapy was introduced. This patient is still in second remission (Table 2).



Discussion



The study showed an example of rare types of acute leukaemia. The description of these cases suggested the requirement of additional tests including cytoplasm staining in cases with atypical immunophenotype in screening routine assay, the warranty of atypical immunophenotype of leukaemia cells in children with other either genetic or immune defects [1, 6, 9]. The very rare combination of determinants on leukaemia cells had to be proven with double staining and specific controls as well as careful gating of the suspected population. The classification of immunophenotype obtained from flow cytometry should be based on acquisition of adequate number of events, low background of unspecific staining and staining of cells without any doubts as compare to isotypic control. The very low intensity of fluorescence understood as low expression of determinants on the surface was difficult for the analysis and suggested the repeating of staining procedure [5, 6, 11]. In AMLL and bi-lineage or bi-phenotypic forms of leukaemia the immunophenotype of leukaemia cells should be monitored during the time of reaching the remission and/or in relapse. The origin of leukaemia cells was confirmed with presence of lineage-restricted determinants in the cytoplasm of cells.
The hypothesis of transformation of pluripotent haematopoietic cells which may normally co-express myeloid and lymphoid antigens prior to commitment to a single lineage or hypothesis of disturbed differentiation of previously normal stem cells could explain the atypical expression of determinants on the surface of leukaemia cells [1,8,15]. The first hypothesis is based on observation of neonatal cells in normal bone marrow expressing myeloid, lymphoid T and B determinants [1, 5, 15].
The atypical expression of determinants is frequently associated with high expression and activity of multidrug resistance molecule (MDR, p-gp) [8, 16-20], expression of CD34 [20] and genetic abnormalities and aberrations. The most common genetic aberrations are:

- fusion gene bcr/abl or Philadelphia chromosome t(9;22)(q34;q11) [1, 3, 8, 16, 21-22],

- rearranged genes (especially in MLL gene) - 11q23 (t(4;11)(q21;q23) [1, 3, 8, 10, 16, 23] and t(9;11)(p21-22;q23) [8, 23-24]

- t(2;7), t(9;12), t(7;12) [8],

- monosomy of chromosome 7 [15],

- trisomy of chromosome 8 [25].

The expression of p-gp studied in a few of our patients was high (more than 90% of blast cells). The expression of p-gp on leukaemia cells with the atypical immunophenotype was frequently observed [16-17, 19-20]. It supported a hypothesis of disturbances in development of stem cell because of the physiological expression of p-pg on this cell [15]. The clinical significance of atypical immunophenotype of leukaemia cells still is under discussion. Atypical expression of determinants and expression of p-gp and CD34 were associated with lower sensitivity to therapy and in consequence, poorer prognosis. In the group of patients with atypical immunophenotype of leukaemia cells the lower percentage of complete remissions and 4 years EFS was observed comparing to a group of patients with typical immunophenotype of leukaemia cells [8, 16, 22]. Moreover, the poorer prognosis was associated with numerous chromosomal aberrations what was the common feature of leukaemia with atypical immunophenotype [1, 3, 8, 10, 21-23]. However, there were some observations that atypical immunophenotype was associated with higher sensitivity to therapy than one-lineage leukaemia [7, 26]. The myeloid co-expression in lymphoid leukaemia was associated with changes of the biology of leukaemia cells close to myeloid with the decrease of the sensitivity to therapy. The opposite results were noted in acute myeloid leukaemia with co-expression of lymphoid determinants [9, 17, 26].
The changes of immunophenotype of leukaemia cells during the course of disease (in early or late relapse) particularly close to mixed lineage leukaemia might suggest the existence of the small population of cells with atypical immunophenotype seen at diagnosis. These cells were resistant to therapy and proliferated as the relapsing population with different immunophenotype than initial. The commonly accepted cut-off point for expression of determinants on the surface of leukaemia cell was 20% of cells what led to disregard of these small subpopulations during routine diagnosis and classifications. In the monitoring of cases with atypical immunophenotype of leukaemia cells the additional analysis during the time of reaching the remission might be helpful as the sensitive leukaemia cells have been eradicated and the more resistant cells were present. The slow disappearing of leukaemia cells during the one-drug (steroid) phase of therapy could be the next signal for additional analysis of immunophenotype of leukaemia cells. The monitoring of leukaemia cells with atypical immunophenotype might be helpful in modification of therapy according to the individual characteristics of leukaemia cells (“the patient tailored therapy”).
Moreover, the most precise description of leukaemia cells including atypical phenomena (aberrant expression of determinants, genetic aberrations) was important for determination of minimal residual disease in complete remission or in time of cessation of therapy. Moreover, the precise description of atypical immunophenotype indicating involvement of stem cells might suggest increased risk of relapse after autoBMT. This atypical immunophenotype suggesting stem cell involvement could explain high rate of relapses after autoBMT in AML with immunophenotype of early precursors (AM0L - AM1L). The other group with possibility of stem cell involvement was acute leukaemia with switch of immunophenotype of leukaemia cells in relapse toward to mixed lineage leukaemia. The genetic study would be very helpful in such cases. For these patients the alloBMT with graft versus leukaemia effect would be more successful therapy.
The increasing percentage of atypical forms of immunophenotype of leukaemia cells observed in flow cytometry under routine procedure suggested including the wide range of techniques into diagnosis of acute leukaemia. The complex diagnosis based on morphology, cytogenetic and immunophenotyping as a routine would allow to classify the most of acute leukaemia including the rare forms. Moreover, the simultaneous use of techniques of rapid evaluation (flow cytometry, PCR, RT-PCR, FISH and others) helped in precise diagnosis and classification of acute leukaemia suggesting the individual modification of therapy for better results.
The frequency of atypical and rare forms of leukaemia is higher in teenagers, children with other defects (immunodeficiency, Down’s syndrome, chromosomal aberrations). The diagnosis of patients from these groups should be especially precise and complex and based on different methods. The complex and extensive diagnosis of leukaemia in children and adults is postulated. The better diagnosis offers the opportunity to choose the most appropriate treatment that would improve the results of therapy.



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Correspondence: Anna Pituch-Noworolska, MD PhD, Medical College, Jagiellonian University, Department of Clinical Immunology,
265 Wielicka st, 30-663 Cracow, Poland. Phone/fax number:: +48 12 658 97 30, e-mail: mipituch@cyf-kr.edu.pl