Multiparameter flow cytometry in the diagnosis and management of acute leukemia.
GENERAL CONSIDERATIONS IN THE APPLICATION OF FLOW CYTOMETRY TO LEUKEMIA DIAGNOSIS
Despite the increasing importance of molecular and genetic features in the subclassification of acute leukemias, morphologic and immunophenotypic analysis remain the primary modalities by which these diseases are first evaluated. It is currently impractical to perform a comprehensive, undirected molecular analysis of hematolymphoid malignancies owing to the myriad different genes involved, but an initial rapid interpretation of morphologic and immunophenotypic data can efficiently direct a narrow and specific search for molecular lesions. Thus, flow cytometry serves as a tool to triage cases for recurrent molecular and genetic testing. In addition, molecular assays may require a high level of technical expertise, available only in specialized reference laboratories, necessitating prolonged specimen processing and analysis times, which may be incompatible with timely clinical management of acute leukemia. By contrast, flow cytometric analysis can be completed within a matter of hours on virtually any case and is often sufficient, when combined with conventional morphologic review, to arrive at a narrow differential or even definitive diagnosis.
For example, a new diagnosis of acute myeloid leukemia lacking expression of CD34 and HLA-DR raises the possibility of an acute promyelocytic leukemia and would prompt careful morphologic review, consideration of cytogenetic fluorescence in situ hybridization (FISH) for the t(15;17); PML-RARA, and clinical evaluation of the patient for evidence of disseminated intravascular coagulation. It should be emphasized that, as a general rule, a specific diagnosis should not be based solely on immunophenotypic findings without morphologic and preferably molecular/genetic correlation.
A wide range of specimens are amenable to flow cytometric evaluation, although in general, the best results are achieved with fluid-based specimens such as peripheral blood and bone marrow aspirates. These specimens are routinely subjected to red blood cell lysis with ammonium chloride as a first step in processing. The remaining leukocyte population is then resuspended in cell culture medium and subjected to a cell count, so that controlled numbers of cells can be submitted for staining with antibodies conjugated to a variety of fluorochromes. Aliquots of the processed cells are then added to individual "tubes" for staining with panels of antibodies. For best results, 500 000 to 1 million cells are stained per tube, although as few as 50000 cells may yield useful results, especially if the neoplastic clone is a prominent component of the specimen. On extremely paucicellular specimens there will be a practical limit to the number of tubes--and hence antigenic markers--that can be analyzed. Finally, the samples are scanned by the flow cytometry instrument, and the resultant data histograms saved for evaluation. Biopsies from solid organs, including bone marrow, undergo a similar process after the addition of a manual disaggregation step and coarse filtration of large particles. Even with careful processing, biopsy specimens, particularly from fibrotic organs such as skin, generally produce specimens with reduced viability and greater distortion of cellular percentages. Even in a nearly ideal specimen, such as a high-quality marrow aspirate, flow cytometry should not be the sole arbiter of blast percentages, as processing may introduce artifacts due to differential susceptibility of cells to degradation during processing and variability in the efficacy of the erythrocyte lysis step. Certain cell types such as megakaryocytes and, to a lesser extent, plasma cells, are selectively vulnerable to loss during processing.
In general, it is preferable to report flow cytometric findings by describing patterns of antigen expression on any aberrant population as well as an estimated percentage of all cell types identified, both aberrant and normal. Simple listings of percentages of antigen positivity for an entire sample represent underutilization of the technology and are less likely to be diagnostic in complex specimens consisting of a small neoplastic population in a reactive background, as in a posttreatment specimen. For data analysis, most laboratories use a "gating" system in which predetermined combinations of cellular characteristics, for example, intrinsic light scatter properties combined with level of CD45 (leukocyte common antigen) expression, are used to define populations of cells that are then characterized with respect to expression of specific antigens. A smaller number of laboratories use a "cluster analysis" process, whereby the raw data from each tube of cells stained with an antibody combination are subjected to manual review by a pathologist, who uses a combination of expected cellular characteristics (similar to gating), combined with visual inspection, to isolate all unique cellular populations. Both methods of analysis can yield similar results if correctly implemented, although cluster analysis is more sensitive when multiple or only small neoplastic populations are present. However, this benefit requires additional effort during analysis, which may not be practical in all settings. Currently, there is a broad range in the number of antibodies per analysis tube used by clinical laboratories, with most using either 3, 4, or 5 "colors" (ie, different fluorochromes). Forward scatter (cell size) and orthogonal/side scatter (cytoplasmic complexity) provide 2 additional analytic parameters. Panels using as many as 10 colors are coming into clinical use and may offer improved sensitivity in recognizing subtly aberrant immunophenotypes, low numbers of neoplastic cells in minimal residual disease analysis, and studies of low-cellularity samples in which only 1 or a few tubes may be analyzed. (3,4)
A large and ever-increasing number of specific, monoclonal antibodies are available for use in flow cytometry. Laboratories will select different panels of markers on the basis of prior experience and personal preference. Nevertheless, there is a subset of markers that are particularly well characterized and broadly useful, forming the core of most immunophenotypic panels. A selection of these markers is listed in Table 2, along with their expected expression patterns in normal bone marrow specimens submitted for flow cytometry.
Only a few antigens are narrowly restricted to a single cell type (lineage specific); most show more complex reactivity. At one extreme, expression of CD3 virtually defines T lymphocytes. By contrast, CD5 is predominantly associated with T cells but is also seen on a small subset of B-lineage cells, and CD2 and CD7 are seen on both T and natural killer cells. At the opposite end of the spectrum, markers such as CD38, HLA-DR, and CD45 are broadly expressed across a wide range of cell types, but they remain crucial to analysis owing to complex but reproducible patterns of expression during maturation, as well as their propensity to be overexpressed or underexpressed in neoplastic populations. The specific immunophenotypic signature of the abnormal cells, as established at diagnosis, will be useful for follow-up after treatment and detection of minimal residual disease, as discussed below.
In a typical case, the presence of immature "blast" cells in peripheral blood or bone marrow initiates a request for flow cytometric analysis. The first step would be to confirm the presence of blasts and preliminarily assign a lineage to them. This may be accomplished through a screening panel as shown in Figure 2. Depending on the phenotype of the cells, further testing is performed and a specific diagnosis or differential is rendered. Selected panels of antibodies used in our laboratory are given in the legend of Figure 2. The number of tubes and the specific antibodies will vary among laboratories, depending on preference and experience. Some laboratories will choose to initially perform a complete marker panel, reducing turnaround time, as compared to this staged process.
ACUTE MYELOID LEUKEMIAS WITH RECURRENT GENETIC ABNORMALITIES
Many of the acute myeloid leukemias in this category do not have distinct immunophenotypic features, as they have been defined by recurrent genetic and molecular changes, which may be associated with a range of morphologic and immunophenotypic features. Nevertheless, some subtypes have a characteristic immunophenotypic fingerprint which, when present, should prompt directed evaluation for the suspected genetic lesion.
[FIGURE 1 OMITTED]
Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1
Acute myeloid leukemia with recurrent t(8;21) generally shows granulocytic differentiation with maturation, and no specific immunophenotypic or morphologic evidence of monocytic, erythroid, or megakaryoblastic differentiation. By flow cytometry, common granulocytic/neutrophilic lineage-associated antigens are expressed including CD13, CD15, CD33, and myeloperoxidase. The blasts are often bright positive for CD34 and may express terminal deoxynucleotidyl transferase (TdT). In addition, these leukemias frequently show expression of B lineage-associated markers, especially CD19, with PAX5 and CD79a less commonly seen (representative example in Figure 3, a). In cases of acute myeloid leukemia showing strong expression of CD19, it is important to evaluate for t(8;21), as this recurrent cytogenetic abnormality supersedes a diagnosis of mixed phenotype acute leukemia (discussed below). If a satisfactory conventional cytogenetic analysis is not available, remaining materials from the flow cytometry specimen may be sent directly for interphase FISH. Some cases show expression of CD56, which may be associated with a worse prognosis. (5) Additional cytogenetic abnormalities, most frequently loss of chromosome 9q, may be present.
Acute Myeloid Leukemia With inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11
Acute myeloid leukemia with recurrent inv(16) or t(16;16) characteristically shows myelomonocytic differentiation with a variable expansion of eosinophils, which may have unusually large cytoplasmic granules with violet coloration. Flow cytometry may identify distinct aberrant blast and myeloid/monocytic populations. Expression of CD14, CD4, CD11b, or combined CD36/CD64 by flow cytometry suggests monocytic differentiation. Although most easily noted by morphologic evaluation, the eosinophils are usually also evident by flow cytometry as a distinct population of cells with high cytoplasmic complexity (side scatter) similar to mature neutrophils, but which appear slightly smaller (less forward scatter) as well as being uniformly CD162.
Acute Promyelocytic Leukemia With t(15;17)(q22;q12); PML-RARA
Corresponding to the French-American-British morphologic category of acute myeloid leukemia M3, acute promyelocytic leukemia with recurrent t(15;17) consists of a neoplastic granulocyte population at the promyelocyte stage of maturation. Normal myeloblasts express CD34, HLA-DR, and CD117 but lose expression of CD34 and HLA-DR with maturation to the promyelocyte stage. The classic hypergranular form of acute myeloid leukemia with t(15;17) frequently shows immunophenotypic features reflective of this pattern, with neoplastic "blasts" that are [CD34.sup.-], HLA-[DR.sup.-], and CD117+ (Figure 3, b). In addition, compared to normal myeloblasts, the neoplastic cells frequently show a marked increase in orthogonal scatter, as might be expected given their cytoplasmic complexity. The microgranular variant more commonly shows at least partial expression of CD34 and HLA-DR and may be positive for CD2. A new diagnosis of acute myeloid leukemia with [CD34.sup.-], HLA-[DR.sup.-] blasts should prompt careful morphologic review and/or consideration of rapid testing for PML-RARA (eg, by FISH) to rule out acute promyelocytic leukemia owing to the increased risk of potentially fatal disseminated intravascular coagulation. However, it should be noted that many [CD34.sup.-], HLA-[DR.sup.-] acute myeloid leukemias will not have an associated PML-RARA1, instead representing clinically and molecularly distinct entities, such as the so-called acute myeloid leukemia with cuplike nuclear invagination. (6)
[FIGURE 2 OMITTED]
Acute Myeloid Leukemia With t(1;22)(p13;q13); RBM15-MKL1
Acute myeloid leukemia with recurrent t(1;22) generally shows evidence of megakaryocytic differentiation, which can be demonstrated by using antibodies targeting CD41 (glycophorin IIb) and CD61 (glycophorin IIIa). While these markers have good specificity, false-positive expression may be seen owing to adherence of platelets to blasts. Comparison to other cell populations may be helpful; for example, if mature granulocytes in the same specimen show apparent expression of CD41 and CD61, then similar staining on the blast population should be regarded with skepticism. Marrow fibrosis may cause difficulty in aspirating neoplastic cells, resulting in marked underestimation of blast percentage by flow cytometry. The blasts are often negative for CD34, HLA-DR, and CD45.
Additional Categories of Acute Myeloid Leukemia With Recurrent Genetic Abnormalities
The remaining examples of acute myeloid leukemia with recurrent genetic abnormalities show less distinctive immunophenotypic features. Acute myeloid leukemia with recurrent t(9;11)(p22;q23); MLLT3-MLL and acute myeloid leukemia with mutated NPM1 frequently, but not invariably, are associated with monocytic differentiation; the latter usually has [CD34.sup.-] blasts. A subset of acute myeloid leukemias with NPM1 mutations has been reported to show an unusual immunophenotype with strong expression of CD33 and myeloperoxidase but minimal expression of other myeloid and monocytic antigens. (7) This group is apparently distinct from the better-known subset of acute myeloid leukemias with "cuplike" nuclear invaginations, which have both NPM1 and FLT3-ITD mutations, and are also [CD34.sup.-], HLA-[DR.sup.-], and myeloperoxidase+ but show less restricted myeloid antigen expression patterns. (6,8)
[FIGURE 3 OMITTED]
Acute myeloid leukemia with inv(3)(q21;q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 may show granulocytic, myelomonocytic, or more rarely, megakaryocytic differentiation. Acute myeloid leukemia with t(6;9)(p23;q34); DEK-NUP214 and acute myeloid leukemia with mutated CEBPA generally show nonspecific granulocytic or myeloid differentiation, although monocytic features may also be present.
ACUTE MYELOID LEUKEMIA NOT OTHERWISE SPECIFIED
A subset of acute myeloid leukemias are not currently assigned to a distinct molecular/genetic subtype, nor do they meet criteria for inclusion in one of the additional distinct World Health Organization acute myeloid leukemia categories described below. These cases are grouped by combined morphologic, cytochemical, and immunophenotypic features into categories reminiscent of the prior French-American-British system. Results of flow cytometric analysis support the morphologic impression, but immunophenotyping is perhaps more important to evaluate for unexpected antigen expression, which might prompt recategorization, for example, expression of lymphoid markers indicative of a mixed phenotype acute leukemia.
By definition, acute myeloid leukemia with minimal differentiation does not show definitive evidence of myeloid differentiation by morphologic or routine cytochemical methods. A comprehensive immunophenotypic evaluation is crucial in these cases to assign lineage to the neoplastic population and rule out a leukemic presentation of a mature lymphoma process. Antigens normally expressed on myeloid blasts are frequently present including CD34, CD117, and HLA-DR; TdT expression is also not uncommon. Pan-myeloid antigens such as CD13 and CD33 are present, but markers normally restricted to maturing myeloid cells such as CD15, CD36, and CD64 are typically absent. Although cytochemical stain results for myeloperoxidase are negative, limited expression may be detected by flow cytometry. B- and T-lineage markers are absent, but CD7 may be seen in the absence of CD3. Acute myeloid leukemia without maturation, acute myeloid leukemia with maturation, and the blasts of acute panmyelosis with myelofibrosis show nonspecific features, with frequent expression of markers associated with myeloid immaturity, such as CD34 and CD117, combined with variable expression of more general markers of myeloid lineage, including CD13, CD15, CD33, and CD64. The rare cases of acute basophilic leukemia reported have frequently shown expression of common myeloid antigens such as CD13 and CD33, as well as CD9, CD11b, and CD123. (9)
In keeping with the morphologic impression, acute myelomonocytic leukemia and acute monoblastic or monocytic leukemias display immunophenotypic evidence of monocytic differentiation. In the former, there may be distinct populations of immunophenotypically aberrant blasts not showing definitive evidence of monocytic differentiation, as well as a population of cells demonstrating expression of CD14, coexpression of CD36 and CD64, or bright expression of CD33, CD4, CD11b, or CD11c. By contrast, acute monoblastic and monocytic leukemias usually show a single population of aberrant cells with evidence of monocytic differentiation, which are usually CD342 and may be [CD117.sup.-]. A recent study (10) has suggested that bright expression of CD64 shows strong correlation with this latter group of leukemias.
Although morphologic examination will generally suggest the possibility of an acute myeloid leukemia with erythroid or megakaryocytic differentiation, this information may not be available to the flow cytometry laboratory, and in some cases the morphologic features are not typical. Moderate to bright expression of CD36 is common for acute myeloid leukemias with erythroid or megakaryocytic differentiation, and when present, may prompt additional studies to evaluate this possibility. CD41 (glycoprotein IIb) and CD61 (glycoprotein IIIa) are strongly associated with megakaryocytic differentiation, but care must be taken to rule out artifact due to platelets adhering to the neoplastic cells, as described above for acute myeloid leukemia with recurrent t(1;22). In general, if platelet adherence is a problem, there will also be similar findings with 1 or more nonblast populations, such as mature granulocytes or monocytes, and comparison may be helpful. Although CD42 (glycoprotein Ib) is less frequently expressed in acute leukemia, it offers strong evidence of megakaryocytic differentiation. Moderate to bright expression of CD71 (transferrin receptor) and glycophorin A (CD235a) provides evidence of erythroid differentiation, but it should be noted that dim expression of the former is seen on many activated cell types, including neoplastic myeloblasts.
UNIQUE CATEGORIES OF ACUTE MYELOID LEUKEMIA
Three subtypes in this group--therapy-related acute myeloid leukemia, acute myeloid leukemia with myelodysplasia-related changes, and myeloid sarcoma--are defined predominantly on the basis of patient history, morphologic and cytogenetic findings, or anatomic location. All 3 frequently have morphologic and immunophenotypic features of a myelomonocytic, monocytic, or monoblastic leukemia. However, a range of immunophenotypes may be seen and do not usually impact diagnostic categorization, as the other features supersede the immunophenotypic findings. However, complete characterization should be performed and reported to support the diagnosis and aid in monitoring of response to therapy.
Myeloid leukemia associated with Down syndrome often shows megakaryocytic features including expression of CD36, CD41, and CD61. The most important differential is generally with transient abnormal myelopoiesis, which has a similar phenotype, although expression of CD11b and CD13 is more common in the leukemia form. (11) Clinical and cytogenetic correlation, including the patient's age, is crucial in this diagnosis.
Blastic plasmacytoid dendritic cell neoplasm most frequently involves the skin, and therefore is less likely to be submitted for flow cytometry as an initial diagnosis. However, staging evaluation does reveal peripheral blood or bone marrow disease in most cases, and in this context, flow cytometry is frequently helpful, especially when involvement of these sites is limited. (12) This neoplasm characteristically expresses CD4, CD45RA, CD56, CD123 (bright), and HLA-DR. There is minimal expression of Blineage, T-lineage, or myeloid-specific markers, with CD7 and CD33 being most common. (12) Results with CD34 and CD117 are usually negative, but results with TdT may be positive. Careful correlation with clinical, morphologic, immunohistochemical, and cytochemical features is required to avoid erroneous diagnosis of this rare entity.
Monitoring Minimal Residual Disease in Acute Myeloid Leukemia
Current chemotherapy protocols are highly effective in producing clinical remission in patients diagnosed with acute myeloid leukemia, but most will eventually have a relapse. (13,14) Cytogenetic and molecular characteristics stratify a proportion of patients into low- and high-risk groups, but most lack definitive prognostic features, resulting in classification as "standard" risk. For these patients, presence of minimal residual disease after chemotherapy, defined as the persistence or recurrence of neoplasia at a level not recognizable by conventional morphologic review, has been shown to have prognostic significance in both adults (15,16) and children. (17) These results may be used to tailor postinduction therapy, (15,18) as the likelihood of relapse in patients with and without minimal residual disease after induction chemotherapy may vary by as much as 5-fold. (17) Furthermore, early reductions in peripheral blood blast counts have been shown to correlate with eventual clinical remission and disease-free survival. (19) Two major modalities have been used to measure minimal residual disease in patients with acute myeloid leukemia: molecular testing and multiparameter flow cytometry. (20) The former is highly sensitive for those patients with recurrent genetic abnormalities detectable by polymerase chain reaction, such as t(15;17), t(8;21), and inv(16). (14,20) An advantage of flow cytometry is that it provides a direct quantification of viable leukemia cells as a proportion of total leukocytes. (18) In those cases in which both flow cytometry and molecular analyses of minimal residual disease are informative, there has been overall concordance, although the sensitivity of the 2 techniques is highly variable, depending upon the molecular and immunophenotypic features of the diagnostic specimen. (21) However, at the current time, most acute myeloid leukemia cases are not amenable to molecular monitoring of minimal residual disease.
By contrast, multiparameter flow cytometry may identify an abnormal/aberrant "leukemia-associated immunophenotype" in more than 90% of cases, if a broad spectrum of monoclonal antibodies is used. (13,14,22) Aberrancies include absence of expected antigens (eg, loss of CD13 or CD33), overexpression of antigens (eg, bright expression of CD33), asynchronous expression of antigens (eg, simultaneous coexpression of immature and mature markers like CD34 and CD11b), lineage infidelity (eg, expression of CD19), and more rarely, aberrant light scatter properties (eg, blasts with increased orthogonal scatter). Aberrant expression of CD2, CD7, CD11b, CD19, and CD56 has been reported as the most helpful. (22) The lower limit of sensitivity is affected by several parameters including sample quality, number of cells analyzed, the degree of immunophenotypic aberrancy, and the technical expertise of the testing laboratory. Ideally, levels of leukemic cells as low as 0.01% may be recognized. Because of the importance of minimal residual disease detection, a thorough immunophenotypic characterization should be performed on all new acute myeloid leukemias even if the flow cytometric results are not required for the initial diagnosis, (20) as subtle immunophenotypic features may be crucial to distinguish persistent disease from reactive changes related to therapy. This immunophenotype should be explicitly documented in the flow cytometry report, as patients frequently receive follow-up treatment in different centers from those where their conditions were originally diagnosed. Rarely, no overt immunophenotypic abnormality will be present at diagnosis, and in these cases, the sensitivity of flow cytometric evaluation for minimal residual disease may be greatly reduced. An additional complicating factor is antigenic shift, (13,20) although the number of cases in which immunophenotypically aberrant blasts convert to an entirely normal pattern of antigen expression is probably low.
As for acute myeloid leukemia, immunophenotypic evaluation of lymphoblastic leukemia/lymphoma (formerly known as acute lymphoblastic leukemia) is essential to confirm the diagnosis and perform further subclassification. With the exception of Auer rods, which when present, unequivocally indicate a myeloid lineage, there is no definitive morphologic feature distinguishing acute myeloid from lymphoblastic leukemia. In addition, there is no reliable morphologic criterion by which B-lineage and T-lineage lymphoblastic leukemias/lymphomas can be distinguished. This latter subdivision is important, as lineage does provide meaningful clinical information, which impacts both prognostic and therapeutic considerations. (23) After lineage has been established, clinical features, such as peripheral leukocyte count and patient age, as well as genetic features including DNA ploidy, stratify patients into different risk groups. Although morphologic variants of lymphoblastic leukemia/ lymphoma do exist, in general, cytologic features do not correlate well with prognostic or specifically known molecular/genetic lesions. Some recurrent genetic lesions are associated with particular immunophenotypic features, however.
Most cases of B-lymphoblastic leukemia/lymphoma show strong expression of multiple B-lineage markers including CD19, CD22, and CD79a. Expression of CD10, often somewhat brighter than for normal immature B-lymphocytes (hematogones), is classic but neither specific nor required for diagnosis. PAX5 and TdT are also commonly seen, while expression of leukocyte common antigen (CD45), CD34, and CD20 may show any pattern of expression from bright to negative (see Figure 3, c for a representative example). Surface immunoglobulin is very rarely present, even in cases with immunoglobulin gene rearrangement. Inappropriate expression of myeloid-lineage markers, especially CD13 and CD33, and less frequently, CD15, is common, (24) but myeloperoxidase positivity would indicate a mixed phenotype acute leukemia. In some cases, the B lymphoblasts do not show aberrant gain and loss of any single antigen, but instead show only an abnormal or "asynchronous" pattern of marker coexpression. For this reason, careful evaluation of the pattern of marker expression may be crucial, particularly in a regenerating posttreatment bone marrow, as normal B-lymphocyte precursors (hematogones) may be increased in these samples. In particular, normal hematogones are initially [CD10.sup.+], CD22(dim+), [CD34.sup.+], and [TdT.sup.+], losing expression of CD34 and TdT before acquiring expression of CD20, and finally losing expression of CD10 only as they develop into mature B lymphocytes with surface antibody expression. B-lymphoblastic leukemia may show a similar spectrum of antigen expression, but often with a more restricted pattern and derangement of the normal maturational sequence. For example, individual neoplastic lymphoblasts may simultaneously express CD34 and CD20, which would not be observed in normal maturing hematogones.
B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities generally conforms to the pattern of CD marker expression described above, although some subtypes show reproducible but not absolute differences in marker profiles. Perhaps the most distinctive are examples associated with t(v;11q23); MLL rearranged, which are often [CD10.sup.-] and [CD15.sup.+], particularly when t(4;11)(q21;q23); AF4-MLL is present. The aberrations t(9;22)(q34;q11.2); BCR-ABL1 and t(12;21)(p12;q22); TEL-AML1 also show expression of myeloid-associated antigens more frequently than most B-lymphoblastic leukemias, especially antigens CD13 and CD33. B-lymphoblastic leukemia with recurrent t(1;19)(q23;p13.3); TCF3-PBX1 is reportedly associated with absence of CD34 as well as expression of CD9. (25) The remaining forms of B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities lack known distinguishing immunophenotypic features.
Although recurrent genetic abnormalities are known in T-lymphoblastic leukemia/lymphoma, their role in prognosis is less clear than for B-lymphoblastic leukemia/ lymphoma and currently distinct categories are not recognized in the World Health Organization classification. Moderate to bright expression of CD3, either on the cell surface or within the cytoplasm, is considered the most lineage-specific marker of T-cell differentiation. In addition, other T lineage-associated markers including CD1a, CD2, CD4, CD5, CD7, and CD8 are variably expressed; it would be rare to see expression of CD3 in isolation. Terminal deoxynucleotidyl transferase, CD34, and CD1a each suggest an immature T-lymphoid process, with TdT the most likely to be present (Figure 3, d). CD10, CD79a, and rarely, PAX5, may be positively expressed and individually do not disprove a diagnosis of T-lymphoblastic leukemia/ lymphoma. Myeloid antigens, especially CD13 and CD33, may also be seen. More rarely, CD117 expression is present and has been associated with FLT3-activating mutations. (26) In adults, expression of CD1a and absence of CD13 have been associated with improved survival. (27) Although it is possible in some cases to further correlate the neoplastic phenotype with a stage of normal thymocyte differentiation, this categorization has only inconsistently been correlated with prognosis, and a recent study (27) suggests it is of limited significance in adults.
Monitoring of Minimal Residual Disease in Lymphoblastic Leukemia/Lymphoma
Detection of minimal residual disease in lymphoblastic leukemia/lymphoma has similar implications as in acute myeloid leukemia, correlating with a worse prognosis, especially when present at the end of consolidation. (20,28) In addition, early treatment responses (blast reduction) also predict remission status after induction as well as likelihood of clinical relapse after therapy. (29,30) Pediatric studies (28) have demonstrated that it is not only the presence of blasts that is important in minimal residual disease, but also their absolute percentage, with levels as low as 0.1% to 0.01% correlating with increased risk of relapse. As significant numbers of T-lineage precursor cells are not normally present in the bone marrow, the identification of T-lineage cells that are [CD34.sup.+] or [TdT.sup.+] at this site strongly suggests residual disease. This is in contrast to B-lineage precursors (hematogones). In fact, hematogones are frequently increased in regenerating, posttherapy bone marrow, but they do retain their characteristic immunophenotypic features. (31) Therefore, the presence of immature B-lineage precursors is not necessarily indicative of persistent/recurrent B-lymphoblastic leukemia. Careful evaluation for immunophenotypic aberrancy, including an abnormal pattern of maturation, is crucial and will be diagnostic in most cases. Newer markers, such as CD81, may also prove helpful in this distinction. (32) A significant fraction of recurrent lymphoblastic leukemias/lymphomas will show an immunophenotypic shift, and therefore it is important that minimal residual disease panels not be too narrowly restricted. (33,34)
Multiple studies have shown correlation between flow cytometric and molecular genetic studies of minimal residual disease in lymphoblastic leukemia/lymphoma, the latter predominantly by real-time quantitative polymerase chain reaction detection of B- and T-cell gene rearrangements or recurrent genetic translocations. (35-37) However, the utility and sensitivity of these 2 modalities for any specific patient may vary. (35,36,38) Although molecular studies with patient-specific oligonucleotide probes are generally considered more sensitive than flow cytometry, the creation and application of these probe sets require molecular expertise. In addition, subsets of patients are not amenable to polymerase chain reaction-based methods owing to the absence of a specific, detectable gene rearrangement or ongoing mutation in the variable regions of the immunoglobulin or T-cell receptor genes. (23,28,37) It has been suggested that the most sensitive strategy may be to apply both flow cytometry and molecular studies to assess minimal residual disease. (35)
ACUTE LEUKEMIAS OF AMBIGUOUS LINEAGE
The 2008 World Health Organization classification combines the former categories of "bilineal" and "biphenotypic" acute leukemias into the single category of mixed phenotype acute leukemia. In combination with the rare acute undifferentiated leukemias, these neoplasms constitute the acute leukemias of ambiguous lineage. Extensive immunophenotypic analysis is required for evaluation of these neoplasms, as morphologically distinct blast populations may not be evident. In general, a mixed phenotype acute leukemia indicates the presence of either (1) two distinct blast populations meeting criteria for at least 2 of the following entities: B-lymphoblastic leukemia, T-lymphoblastic leukemia, and acute myeloid leukemia; or (2) a single population showing definitive evidence of differentiation toward 2 of these lineages. In this latter case, the World Health Organization provides specific guidelines, that is, myeloperoxidase staining or expression of 2 or more monocytic antigens (CD11c, CD14, CD64, nonspecific esterase, lysosome) indicates myeloid lineage; strong cytoplasmic or surface CD3 expression indicates T lineage; and strong CD19 expression associated with weak expression of CD10, CD22, or CD79a or weak CD19 expression plus strong expression of 2 of the same markers indicates B-lineage. Therefore, at a minimum, all acute leukemias should be assayed for myeloperoxidase, a selection of monocytic markers, CD19, and cytoplasmic/surface CD3 to detect unsuspected cases of mixed phenotype acute leukemia. Currently, there is some ambiguity in the interpretation of these markers, particularly cases in which an apparently lymphoblastic leukemia shows limited expression of myeloperoxidase by flow cytometry not corroborated by cytochemical staining. In the absence of the recurrent genetic abnormalities t(9;22)(q34;q11.2); BCR-ABL1 or t(v;11q23); MLL rearranged, subclassification of mixed phenotype acute leukemias is based upon the lineages present. With the current criteria, the most commonly identified form of mixed phenotype acute leukemia appears to be the combination of B-lymphoid and myeloid differentiation (Figure 3, e).
Ongoing developments in the understanding of hematolymphoid malignancies have resulted in revision to prior classification systems, better reflecting clinically relevant entities that affect prognosis and therapy. Increasingly, acute leukemias are defined by discrete, recurrent genetic abnormalities. Despite these advances, morphologic and immunophenotypic analyses remain as the foundation for initial evaluation, providing a rapid assessment and directing specific molecular genetic tests, which are impractical to apply in a universal or "shotgun" approach. In addition, immunophenotypic data obtained via flow cytometry may directly correlate with prognosis. Finally, as molecular medicine evolves, specific antitumor agents, including monoclonal antibodies to tumor antigens, are transitioning from investigational to clinical use. (39-41) Flow cytometry provides a direct assessment of the varied surface antigen expression on hematolymphoid neoplasms, (42) facilitating a rational and individualized selection of such targeted immunotherapy strategies.
(1.) Swerdlow SH, Campo E, Harris NL, et al, eds. Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2008. World Health Organization Classification of Tumours; vol 2.
(2.) Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114(5):937-951.
(3. Voskova D, Schnittger S, Schoch C, Haferlach T, Kern W. Use of five-color staining improves the sensitivity of multiparameter flow cytomeric assessment of minimal residual disease in patients with acute myeloid leukemia. Leuk Lymphoma. 2007;48(1):80-88.
(4.) Wehner S, Soerensen J, Schwabe D, et al. 10-Parameter flow cytometry as a new tool to improve diagnosis and MRD follow-up of acute leukemias. Klin Padiatr. 2009;221(6):393-395.
(5.) Baer MR, Stewart CC, Lawrence D, et al. Expression of the neural cell adhesion molecule CD56 is associated with shortremission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). Blood. Aug 15 1997;90(4): 1643-1648.
(6.) Chen W, Konoplev S, Medeiros LJ, et al. Cuplike nuclei (prominent nuclear invaginations) in acute myeloid leukemia are highly associated with FLT3 internal tandem duplication and NPM1 mutation. Cancer. 2009;115(23):54-81 5489.
(7.) Kern W, Haferlach C, Bacher U, Haferlach T, Schnittger S. Flow cytometric identification of acute myeloid leukemia with limited differentiation and NPM1 type A mutation: a new biologically defined entity. Leukemia. 2009;23(7):1361-1364.
(8.) Oelschlaegel U, Mohr B, Schaich M, et al. HLA-DRneg patients without acute promyelocytic leukemia show distinct immunophenotypic, genetic, molecular, and cytomorphologic characteristics compared to acute promyelocytic leukemia. Cytometry B Clin Cytom. 2009;76(5):321-327.
(9.) Lichtman MA, Segel GB. Uncommon phenotypes of acute myelogenous leukemia: basophilic, mast cell, eosinophilic, and myeloid dendritic cell subtypes: a review. Blood Cells Mol Dis. 2005;35(3):370-383.
(10.) Dunphy CH, Tang W. The value of CD64 expression in distinguishing acute myeloid leukemia with monocytic differentiation from other subtypes of acute myeloid leukemia: a flow cytometric analysis of 64 cases. Arch Pathol Lab Med. 2007;131(5):748-754.
(11.) Karandikar NJ, Aquino DB, McKenna RW, Kroft SH. Transient myeloproliferative disorder and acute myeloid leukemia in Down syndrome: an immunophenotypic analysis. Am J Clin Pathol. 2001;116(2):204-210.
(12.) Herling M, Jones D. CD4+/CD56+ hematodermic tumor: the features of an evolving entity and its relationship to dendritic cells. Am J Clin Pathol. 2007; 127(5):687-700.
(13.) Buccisano F, Maurillo L, Spagnoli A, et al. Monitoring of minimal residual disease in acute myeloid leukemia. Curr Opin Oncol. 2009;21(6):582-588.
(14.) Al-MawaliA, Gillis D, Lewis I. The role of multiparameter flow cytometry for detection of minimal residual disease in acute myeloid leukemia. Am J Clin Pathol. 2009;131(1):16-26.
(15.) San Miguel JF, Vidriales MB, Lopez-Berges C, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood. 2001;98(6):1746-1751.
(16.) Maurillo L, Buccisano F, Del Principe MI, et al. Toward optimization of postremission therapy for residual disease-positive patients with acute myeloid leukemia. J Clin Oncol. 2008;26(30):4944-4951.
(17.) Sievers EL, Lange BJ, Alonzo TA, et al. Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children's Cancer Group study of 252 patients with acute myeloid leukemia. Blood. 2003;101(9):3398-3406.
(18.) Shook D, Coustan-Smith E, Ribeiro RC, Rubnitz JE, Campana D. Minimal residual disease quantitation in acute myeloid leukemia. Clin Lymphoma Myeloma. 2009;9(suppl 3):S281-S285.
(19.) Lacombe F, Arnoulet C, Maynadie M, et al. Early clearance of peripheral blasts measured by flow cytometry during the first week of AML induction therapy as a new independent prognostic factor: a GOELAMS study. Leukemia. 2009;23(2):350-357.
(20.) Bene MC, Kaeda JS. How and why minimal residual disease studies are necessary in leukemia: a review from WP10 and WP12 of the European LeukaemiaNet. Haematologica. 2009;94(8):1135-1150.
(21.) Kern W, Haferlach C, Haferlach T, Schnittger S. Monitoring of minimal residual disease in acute myeloid leukemia. Cancer. 2008;112(1):4-16.
(22.) Al-Mawali A, Gillis D, Hissaria P, Lewis I. Incidence, sensitivity, and specificity of leukemia-associated phenotypes in acute myeloid leukemia using specific five-color multiparameter flow cytometry. Am J Clin Pathol. 2008;129(6): 934-945.
(23.) Onciu M. Acute lymphoblastic leukemia. Hematol Oncol Clin North Am. 2009;23(4):655-674.
(24.) Seegmiller AC, Kroft SH, Karandikar NJ, McKenna RW. Characterization of immunophenotypic aberrancies in 200 cases of B acute lymphoblastic leukemia. Am J Clin Pathol. 2009;132(6):940-949.
(25.) Borowitz MJ, Hunger SP, Carroll AJ, et al. Predictability of the t(1;19)(q23;p13) from surface antigen phenotype: implications for screening cases of childhood acute lymphoblastic leukemia for molecular analysis: a Pediatric Oncology Group study. Blood. 1993;82(4):1086-1091.
(26.) Paietta E, Ferrando AA, Neuberg D, et al. Activating FLT3 mutations in CD117/KIT(+) T-cell acute lymphoblastic leukemias. Blood. 2004;104(2):558 560.
(27.) Marks DI, Paietta EM, Moorman AV, et al. T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the large randomized prospective trial (UKALL XII/ECOG 2993). Blood. 2009;114(25):5136-5145.
(28.) Borowitz MJ, Devidas M, Hunger SP, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study. Blood. 2008;111(12):5477-5485.
(29.) Basso G, Veltroni M, Valsecchi MG, et al. Risk of relapse of childhood acute lymphoblastic leukemia is predicted by flow cytometric measurement of residual disease on day 15 bone marrow. J Clin Oncol. 2009;27(31):5168-5174.
(30.) Ratei R, Basso G, Dworzak M, et al. Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction. Leukemia. 2009;23(3): 528-534.
(31.) Babusikova O, Zeleznikova T, Kirschnerova G, Kankuri E. Hematogones in acute leukemia during and after therapy. Leuk Lymphoma. 2008;49(10):1935 1944.
(32.) Muzzafar T, Medeiros LJ, Wang SA, Brahmandam A, Thomas DA, Jorgensen JL. Aberrant underexpression of CD81 in precursor B-cell acute lymphoblastic leukemia: utility in detection of minimal residual disease by flow cytometry. Am J Clin Pathol. 2009;132(5):692-698.
(33.) Borowitz MJ, Pullen DJ, Winick N, Martin PL, Bowman WP, Camitta B. Comparison of diagnostic and relapse flow cytometry phenotypes in childhood acute lymphoblastic leukemia: implications for residual disease detection: a report from the children's oncology group. Cytometry B Clin Cytom. 2005;68(1):18-24.
(34.) Chen W, Karandikar NJ, McKenna RW, Kroft SH. Stability of leukemia associated immunophenotypes in precursor B-lymphoblastic leukemia/lymphoma: a single institution experience. Am J Clin Pathol. 2007;127(1):39-46.
(35.) Neale GA, Coustan-Smith E, Stow P, et al. Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia. 2004;18(5):934-938.
(36.) Malec M, van der Velden VH, Bjorklund E, et al. Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping. Leukemia. 2004;18(10):1630-1636.
(37.) Ryan J, Quinn F, Meunier A, et al. Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multipletime-points reveals high levels of concordance between molecular and immunophenotypic approaches. BrJHaematol. 2009;144(1):107-115.
(38.) Szczepanski T. Why and how to quantify minimal residual disease in acute lymphoblastic leukemia? Leukemia. 2007;21(4):622-626.
(39.) Gojo I. Improving treatment strategies for acute lymphoblastic leukemia by combining immunotherapy and chemotherapy. Leuk Res. 2008;32(6):847-849.
(40.) Raetz EA, Cairo MS, Borowitz MJ, et al. Chemoimmunotherapy reinduction with epratuzumab in children with acute lymphoblastic leukemia in marrow relapse: a Children's Oncology Group Pilot Study. J Clin Oncol. 2008;26(22): 3756-3762.
(41.) Griffin TC, Weitzman S, Weinstein H, et al. A study of rituximab and ifosfamide, carboplatin, and etoposide chemotherapy in children with recurrent/ refractory B-cell (CD20+) non-Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia: a report from the Children's Oncology Group. Pediatr BloodCancer. 2009;52(2):177-181.
(42.) Chevallier P, Robillard N, Houille G, et al. Simultaneous study of five candidatetargetantigens(CD20, CD22, CD33, CD52, HER2) for antibody-based immunotherapy in B-ALL: a monocentric study of 44 cases. Leukemia. 2009; 23(4):806-807.
John M. Peters, MD, PhD; M. Qasim Ansari, MD
Accepted for publication September 8, 2010.
From the Department of Pathology, University of Texas Southwestern Medical Center, Dallas. Dr Peters is now with ProPath Laboratories, Dallas, Texas.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: M. Qasim Ansari, MD, Department of Pathology, Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, TX 753909073 (e-mail: firstname.lastname@example.org).
Table 1. World Health Organization (2008) Categories of Acute Leukemiaa Acute myeloid leukemia AML with recurrent cytogenetic abnormalities (see Figure 1) AML with myelodysplasia-related changes Therapy-related myeloid neoplasms AML NOS Morphologic and immunophenotypic subtypes Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukemia of ambiguous lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BRC-ABL1 Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged Mixed phenotype acute leukemia, NOS (subclassify by phenotypes) B-lymphoblastic leukemia/lymphoma B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B-lymphoblastic leukemia/lymphoma NOS T-lymphoblastic leukemia/lymphoma Abbreviations: AML, acute myeloid leukemia; NOS, not otherwise specified. (a) The current World Health Organization classification includes entities defined by recurrent genetic abnormalities, cytomorphology, and clinical and immunophenotypic features. Although acute myeloid leukemias with recurrent genetic abnormalities are not strictly linked to specific morphologic or immunophenotypic characteristics, in many cases an association exists (refer to Figure 1). The unique subtypes of acute myeloid leukemia listed have a variable association with specific morphologic and immunophenotypic features. For example, the myeloid neoplasms associated with down syndrome show a marked relative increase in apparent megakaryocytic differentiation. Table 2. Common Markers Used in Flow Cytometric Immunophenotyping (a) Myelo- Promyelo- Maturing Mono- Antigen blasts cytes Grans cytes Erythroids CD2 CD3 2 2 2 2 2 CD4 2 2 2 2 2 CD5 2 2 2 2 2 CD7 2 2 2 2 2 CD8 2 2 2 2 2 CD19 2 2 2 2 2 CD20 2 2 2 2 2 CD22 2 2 2 2 2 CD79a 2 2 2 2 2 CD13 + + + + 2 CD14 2 2 + ++ 2 CD15 2 + + 2 2 CD33 + + + ++ + CD36 2 2 2 ++ CD117 + + 2 2 + CD64 2 2 + + 2 MPO Sub + + -/+ 2 CD71 2 2 2 2 ++ GlyA 2 2 2 2 ++ CD41 2 2 2 2 2 CD61 2 2 2 2 2 CD10 2 2 + 2 2 CD38 + Var (c) Var + 2 CD45 + + + + 2 HLA-DR + 2 2 + 2 CD34 + 2 2 2 2 TdT 2 2 2 2 2 Megakar- B Lym- T Lym- Antigen yocytes phoid phoid CD2 + CD3 2 2 + CD4 2 2 Sub (b) CD5 2 2 + CD7 2 2 + CD8 2 2 Sub CD19 2 + 2 CD20 2 + 2 CD22 2 + 2 CD79a 2 + 2 CD13 2 2 2 CD14 2 2 2 CD15 2 2 2 CD33 2 2 2 CD36 + 2 2 CD117 2 2 2 CD64 2 2 2 MPO 2 2 2 CD71 2 2 2 GlyA 2 2 2 CD41 + 2 2 CD61 + 2 2 CD10 2 Sub 2 CD38 2 Var Var CD45 + + + HLA-DR 2 + 2 CD34 2 Sub 2 TdT 2 Sub 2 Antigen Comments CD2 LFA-2;pan T-cell marker CD3 OKT3;pan T-cell marker CD4 MHC-II associated; helper T-cells CD5 Leu-1; pan T-cell marker CD7 Leu-9; pan T-cell marker CD8 MHC-I associated;cytotoxic T cells CD19 Leu-12; pan B-cell marker CD20 L26;B-cell marker CD22 BL-CAM; pan B-cell marker CD79a MB-1; pan B-cell marker CD13 Aminopeptidase N; pan myeloid marker CD14 LPS receptor; bright on monocytes CD15 LeuM1; maturing granulocytes CD33 Sialic acid adhesion molecule; pan myeloid marker CD36 GP IIIb/IV CD117 c-kit; bright on mast cells CD64 FC-g receptor MPO Myeloperoxidase; definitive myeloid marker CD71 Transferrin receptor; dim expression on activated cells GlyA CD235a;carries MN antigens on red cells CD41 GP IIb;megakaryocytic CD61 GP IIIa; megakaryocytic CD10 CALLA, also expressed by hematogones CD38 Broadly expressed CD45 Leukocyte common antigen HLA-DR Class II MHC component CD34 Adhesion molecule; marker of immature cells TdT Nucleotide transferase; marker of immature cells Abbreviations: CALLA, common acute lymphoblastic leukemia antigen; GlyA, glycophorin A; GP, glycoprotein; LPS, lipopolysaccharide; MHC, major histocompatibility complex;2, absence of expression on normal bone marrow populations;+, presence of expression on normal bone marrow populations; ++, bright expression. (a) A selection of markers commonly used in the evaluation of acute leukemias is listed. In practice, most laboratories will use a more extensive panel of markers, but not all markers are relevant to or used in all cases. (b) Sub stands for subset, indicating a subpopulation of cells within the lineage showing expression of a particular marker. For example, stage I (early) hematogones are CD34+ but more mature cells in the B-lymphoid lineage are CD342. (c) Var stands for variable, indicating that expression of a marker is variable in ways that are not always predictable.
|Printer friendly Cite/link Email Feedback|
|Author:||Peters, John M.; Ansari, M. Qasim|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Article Type:||Disease/Disorder overview|
|Date:||Jan 1, 2011|
|Previous Article:||Diagnosing emerging and reemerging infectious diseases: the pivotal role of the pathologist.|
|Next Article:||Molecular testing of solid tumors.|