Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section C – Diagnosing Cancer: Pathology and Laboratory Medicine

Chapter 17 – Flow Cytometry in Oncologic Diagnosis

Michael J. Borowitz

SUMMARY OF KEY POINTS

INTRODUCTION

Flow cytometry is used to study attributes of individual cells in a suspension. Over the past two decades, flow cytometry has become increasingly sophisticated; high-speed sorters and analyzers capable of detecting more than a dozen colors simultaneously have kept flow cytometry in the forefront as a tool for the fundamental investigation into cancer. At the same time, flow cytometry has matured from a research technology to one that is part of the routine clinical laboratory. This chapter focuses on diagnostic aspects of flow cytometry.

METHODS

A flow cytometer analyzes large numbers of cells one at a time, making it an ideal tool for examination of the properties of populations of cells. It is complementary to imaging: although it does not provide as much detail about individual cells, it provides much better statistical measurements, and is able to characterize different groups of cells in heterogeneous mixtures. It also can physically sort specifc subpopulations of cells. Although a detailed discussion of the workings of a flow cytometer is outside the scope of this chapter, the chapter begins with a discussion of a few general principles.

Functional Components

A flow cytometer has three separate components: a fluidics system; an optical platform; and signal-processing electronics. The fluidic system aspirates the sample, mixes it with a sheath fluid to produce laminar flow, and conducts the cell suspension past the sensing zone where individual cells are examined. The optical platform consists of the laser light source(s), lenses to focus the light on the passing cell stream, band-pass filters to capture light of restricted wavelengths, and photomultiplier tubes (PMTs) that capture the emitted signals. The electronics convert photons to electrical signals in proportion to the total light contacting the PMTs, and amplify and scale the signals so that data can be readily analyzed. The value of each collected parameter is “quantitized” and assigned to a particular channel. Higher channels reflect brighter signals. Current software packages reprocess collected data electronically in a variety of formats; this flexibility of data analysis is a critical part of what makes this technology useful.

Electronically reprocessed data are displayed by software programs in the form of dot plots or histograms. The most useful displays usually correlate one parameter with another, with each dot representing a single event (i.e., cell) with the x- and y-channel values for the two chosen parameters. In addition to fluorescence, forward scatter and right-angle or side scatter can be displayed. Forward scatter is roughly proportional to the size of the cells, whereas side scatter is a measure of internal cellular complexity, which for hematopoietic cells usually means granularity. Typically, dot plots of either forward versus side scatter, or scatter versus fluorescence, are used to identify populations of interest in a process called gating, and additional displays show additional fluorescence or scatter characteristics of these populations. Because scatter measurements are made parallel to, and independent of, the fluorescence measurements, four-color flow cytometry, for example, is equivalent to six-parameter cytometry. The term multiparameter flow cytometry typically is used to define simultaneous analysis of five or more parameters on individual cells.

Fluorochromes and Fluorescence

Fluorochromes have spectral characteristics that allow them to absorb light of certain wavelengths and then to emit light at longer wavelengths. Emission, a specific characteristic of the compound, is not limited to a fixed wavelength, but, rather, constitutes a spectrum, with variable numbers of photons emitted at different wavelengths. Different fluorochromes, including fluorescein isothiocyanate (FITC) and phycoerythrin (PE) as well as others, all have the capacity of absorbing light at 488 nm but emit it at different wavelengths, thereby making it possible to perform multicolor flow cytometry with a single laser emitting at 488 nm. Tandem conjugates, which covalently couple two fluorochrome molecules, allow emitted light to be transferred from one molecule to the other and can greatly increase the number of colors that can be detected with a single laser. Additional dyes such as allophycocyanin, which emit longer wavelengths, cannot be excited by a 488-nm laser but require a second light source, most frequently one emitting light at 635 nm. The combination of new dyes, more tandem conjugates, and new instruments with even more lasers has made it possible to perform flow cytometry to detect more than a dozen colors, and 6- or even 8- to 10-color flow cytometry is now being performed routinely in clinical laboratories.

APPLICATIONS OF FLOW CYTOMETRY TO CLINICAL ONCOLOGY

Flow cytometry plays a significant role in the diagnosis, classification, and management of patients with acute leukemia, chronic lymphoproliferative disorders, and non-Hodgkin's lymphoma. The early promise of this technology in the management of patients with solid tumors has not been completely realized, but it still plays a role in certain areas.

Acute Leukemia

Flow-cytometric immunophenotyping has become standard in the evaluation of new patients with acute leukemia. The most obvious role of flow cytometry is in distinguishing lymphoid from myeloid leukemia, but flow cytometry can help in the diagnosis and management of these patients in many ways ( Box 17-1 ).

Box 17-1 

Lineage Assignment in Acute Leukemia

Phenotypic analysis of a bone marrow that is completely replaced by blasts is an almost elementary problem. Multiparameter flow cytometry, however, can dissect and categorize all populations in bone marrow, and, most important, can distinguish leukemic cells from normal, even when the leukemic cells are not the majority population. The antibody panel used to study patients with acute leukemia typically contains representative markers of all lineages, with some redundancy to allow recognition and classifcation because many antigens may be aberrantly lost or acquired in leukemic cells. ^{[1] [2]} No standard combinations of antibodies are used by all laboratories, but certain combinations have proved particularly useful not only for the most economical classification of leukemia, but also for their ability to demonstrate characteristic aberrant patterns that may be extremely useful for the detection of residual disease in follow-up samples, as discussed later in this chapter.

Diffculties encountered in the interpretation of flow cytometry data derive largely from two problems. First, in cases in which leukemic cells are not an obviously dominant population, it is important to ensure that the cells of interest are analyzed. The most useful general approach for this takes advantage of the fact that the common leukocyte antigen CD45 is differentially expressed on different types of hematopoietic cells, and, when combined with side scatter, produces a display in which blasts occupy a unique position not occupied by normal cells ( Fig. 17-1 ).^[3] Thus, combining CD45 in one color with multiple combinations of antibodies in additional colors allows detailed characterization of blast populations in marrow even when they are present only in low numbers.

Failure to select the leukemic cell population for analysis, or including a mixture of leukemic and normal cells in a gate, may cause confusion in the reporting of flow cytometry results. It is best to identify the leukemic population visually and to provide a detailed description of the antigens expressed on the leukemic population, especially in myeloid leukemias, in which the dynamic patterns of maturation associated with morphologic variation in AML are reflected in changes in both light scatter and antigen expression as the leukemic cells mature. Tabular arrays of “percentage positive” are not recommended as part of a flow cytometry report, because they cannot reflect this complexity and may cause confusion.^[4]

The second problem in interpreting flow cytometry results in leukemia derives from the fact that most of the reagents used in classifying leukemia are only relatively rather than absolutely specific. Thus, correct classification requires not only a panel with some redundancy, but also an understanding of patterns of reactivity of the antibodies. Markers such as CD13 and CD33, which are considered myeloid antigens because they originally were produced against myeloid leukemia cells, are found in up to 50% of cases of lymphoid leukemia,^[5] and interpretation of leukemias positive for these markers continues to be a cause of confusion. Generally speaking, the most specific markers of a given lineage are not highly sensitive, and the most sensitive ones are not specific. This is true more of myeloid markers than of lymphoid markers, so that lymphoid leukemias usually can be recognized precisely, whereas poorly differentiated myeloid leukemias usually are defined by the presence of myeloid markers in the absence of specific lymphoid antigens.

Acute Leukemias of Indeterminate or Ambiguous Lineage

Although almost all cases of acute leukemia can be categorized easily regarding lineage, the lack of absolute specificity of most markers, and the promiscuity of their expression, implies that some cases cannot be resolved easily. Unfortunately, considerable controversy exists about the use of the terms mixed lineage and biphenotypic leukemia. Leukemias that express myeloid- and lymphoid-associated markers in combination represent a heterogeneous group of diseases. A scoring system has been proposed^[6] that assigns various point values to different antigens, with a diagnosis of “biphenotypic leukemia” rendered if the score is high for more than one lineage. Although this is an objective method of defining lineage, it mixes different kinds of cases with different molecular abnormalities. The scoring system as originally conceived also does not distinguish between cases in which blasts coexpress antigens of different lineages and those in which there are distinct leukemic blast populations. The latter situation is much less common, but can be thought of properly as true mixed-lineage leukemia. Certain of these, such as the combination of β-precursor ALL and AML in patients with the Philadelphia chromosome, represent distinct entities.

Association of Immunophenotype and Molecular Abnormalities

Many phenotypes in both ALL and AML are highly associated with characteristic cytogenetic abnormalities.^[7] In ALL, these include cases associated with MLL rearrangements, TEL-AML1 or E2A-PBX1. In AML, the most important link is in promyelocytic leukemia with the t(15;17). Whereas lack of HLA-DR is the best-known abnormality, only about half of cases of DR-negative AMLs turn out to be acute promyleocytic leukemia (APL), and other combinations of marker expression are much more sensitive and specific for APL.^[8] AML associated with the t(8;21) also shows a characteristic phenotype.^[9]

Minimal Residual Disease Detection in Acute Leukemia

Several recent studies have demonstrated that the presence of residual leukemic cells in the marrow of patients in clinical and morphologic remission is a very strong adverse prognostic factor. ^{[10] [11] [12] [13] [14] [15] [16]} Although the most extensive data exist in childhood ALL, ^{[10] [11] [12]} the principle has also been shown to apply to adult ALL^[13] and to AML. ^{[14] [15] [16]}

Both molecular and flow-based methods have been used to detect minimal residual disease (MRD). Flow-based assays of MRD are based on the principle that nearly all leukemias show a pattern of expression of antigens that is aberrant compared with the pattern seen in normal differentiation. ^{[10] [11] [12] [13] [14] [15]} This aberrancy can take several forms. Some leukemic cells can abnormally express antigens of a different lineage or show loss of expression of a normal lineage marker. A more common finding is expression of normal differentiation antigens, but at an intensity that is different from that expected for a particular stage of differentiation. This latter attribute makes flow MRD analysis applicable to most cases of leukemia. Nonetheless, recognizing these deviations requires a clear understanding of patterns of maturation in normal differentiation, including marrow regeneration, as viewed in multiparameter space.

The pattern of antigen acquisition and loss during β-cell maturation in the bone marrow has been very well characterized, and certain markers are particularly useful for distinguishing normal and leukemic maturation. Consideration of markers including intensity of CD45, CD34, CD10, CD58 and CD38 or aberrant coexpression of myeloid or other unexpected antigens can allow detection of as few as 1 in 10⁴ leukemic cells, even when normal β-cell precursors are present in significant numbers ( Fig. 17-2 ). Marrow T-ALL can also be distinguished from normal T cells, most readily by coexpression of cytoplasmic CD3 and TdT, which is never seen on any normal cell.^[10] Detection of myeloid MRD usually is a more elaborate process because of the greater phenotypic heterogeneity in AML. Certain aberrant combinations, including coexpression of CD34 and CD56, CD117 and CD15, or CD7 and myeloid antigens occur with sufficient frequency to be useful in a large number of cases. ^{[14] [15] [16]} To achieve 10^-4 sensitivity in essentially all cases may require design of custom panels unique to a particular leukemia. Because abnormal populations at diagnosis may not persist at recurrence, monitoring patients with acute leukemia requires following more than one aberrant phenotype.

Myelodysplasia and Chronic Myeloproliferative Disorders

Recently it has become apparent that flow cytometry can detect abnormalities in marrow disorders other than acute leukemia. Patients with chronic myeloproliferative disorders invariably show abnormalities in either blast phenotype or maturation pattern,^[17] although routine clinical phenotyping of MPDs is not performed in most cases. Interest in using flow cytometry as a primary diagnostic modality in myelodysplastic syndromes is increasing. In addition to finding phenotypically abnormal blasts, many studies have demonstrated characteristic phenotypic abnormalities in either myeloid or erythroid maturation in myelodysplastic syndrome (MDS), as well as describing abnormalities in granulocyte light scatter that correspond to the morphologic finding of hypogranularity. ^{[18] [19] [20]}Although a scoring system has been proposed to use flow cytometric characteristics to diagnose MDS,^[20] the sensitivity and specificity of these findings have not been rigidly established, and it is not clear whether flow cytometry significantly improves the accurate diagnosis of early MDS. Nevertheless, it is clear that the technique can add useful adjunctive information to the diagnosis and possibly the prognosis of MDS.

Chronic Lymphoproliferative Disorders and Lymphoma

Lymphoid Tissue Analysis

Flow-cytometric immunophenotyping plays a major role in the evaluation of a patient with lymphoma (see Box 17-1 ). ^{[21] [22] [23]} Lymphoid tissue first must be disaggregated to produce a cell suspension suitable for flow-cytometric analysis. Although this is readily accomplished in most low-grade lymphomas, high-grade lymphomas may give a nonrepresentative sample because of the greater fragility of the neoplastic cells compared with residual normal cells. Fibrosis also may make disaggregation difficult or result in disruption of neoplastic cells. Consequently, flow-cytometric analysis of lymphoma requires careful gating to ensure that the cells of interest are analyzed.

In contrast to the situation with acute leukemias, no single antibody serves as a useful surrogate for the neoplastic population. However, in β-cell malignancies, neoplastic B cells usually greatly outnumber normal cells (although there may be normal T cells), so that use of a pan-B-cell antibody such as CD19 or CD20 can help to isolate the neoplastic cells for analysis. In addition, forward scatter, as a marker of cell size, may be a very useful parameter to help distinguish frequently larger neoplastic cells from smaller normal counterparts.

Although some laboratories prefer tissue immunohistochemistry to flow cytometry for phenotyping lymphomas, the two techniques are complementary. The principle advantages of flow cytometry relate to its speed and its ability to identify and phenotype precisely the neoplastic elements in a heterogeneous sample. A complete phenotypic characterization, and, in many cases, a diagnosis can be achieved within a few hours from the time of biopsy, rather than the several days needed for histopathologic examination and immunostaining. Flow cytometry also is far better than immunohistochemistry for demonstrating light-chain restriction, and therefore clonality, in β-cell neoplasms.

The disadvantages of flow cytometry come from the architectural disruption created by making a cell suspension and the aforementioned possible loss of cells of interest. Hodgkin's lymphoma, in particular, is not diagnosed by routine flow cytometry, although recent studies have demonstrated that careful attention to the procedures used for phenotyping can overcome this drawback.^[24] Even in non-Hodgkin's lymphoma, grading may be difficult from flow-cytometric immunophenotyping alone, and precise classification can be accomplished only rarely; lymphoma classification still relies heavily on morphology, and this must be correlated with flow-cytometric information.

Classification of lymphoma and grading, in particular, can be improved by assessment of DNA content by flow cytometry (as discussed later in this chapter), because S-phase fraction is closely correlated with grade. Burkitt's lymphoma, which has the highest S fraction of any neoplasm, usually can be reliably identifed by flow cytometry. However, in lesions such as follicular lymphoma, grading systems still are based on morphologic criteria, and although there may be reason to expect that flow-based methods might be more objective, these have not been validated in large series to the point at which they are generally accepted.

One of the most fruitful applications of flow cytometry in the diagnosis of lymphoma is in the analysis of specimens from fine-needle aspiration (FNA). ^{[25] [26]} Cells obtained by FNA already are in suspension, and flow-cytometric analysis contributes significantly to the cytopathologic diagnosis of lymphoma, which is notoriously difficult. Although FNA has been used for years in the evaluation of recurrent adenopathy in patients with known lymphoma, it can serve as a primary diagnostic modality in lymphoma when combined with flow cytometry. Most non-Hodgkin's lymphomas can be recognized and graded, and, importantly, lymphomas also can usually be excluded. Optimal application of this technology, however, requires close collaboration between the cytopathologist and flow cytometrist, a clear understanding of the strengths and limitations of the two techniques, and a willingness to revert to open biopsy in ambiguous cases.

Blood and Marrow Analysis

Limitations attendant on generating cell suspensions do not apply to patients with blood or marrow involvement by chronic lymphoproliferative disorders. Subclassification of these lesions relies heavily on flow cytometry. ^{[21] [23] [27] [28] [29]} Chronic lymphocytic leukemia (CLL) is essentially defined by its immunophenotypic characteristics. Other β-cell lymphoproliferative disorders have characteristic, if not always absolutely specific, phenotypes. ^{[21] [23] [27] [28] [30]} Mantle cell lymphoma often can be recognized in its leukemic phase, although the most specific marker, cyclin D1, is very difficult to detect by flow with current techniques. Hairy cell leukemia, conversely, has not only a characteristic but also a highly specific phenotype ^{[23] [31]}; occasionally patients with unexplained pancytopenia can be determined to have hairy cell leukemia when only a tiny number of blood cells with the classic phenotype are identified^[31] ( Fig. 17-3 ).

Recently, considerable attention has been paid to the use of flow cytometry to detect prognostic factors in CLL. Recognizing that CLL can be subdivided into a poor-prognosis type associated with nonmutated immunoglobulin V region genes and a better-prognosis mutated phenotype, several studies have attempted to identify flow surrogates. The first marker found to be prognostic was CD38, although the correlation with either mutational status or prognosis is imperfect. ^{[32] [33] [34]} Zeta-chain-associated protein kinase 70 (ZAP 70) appears to be a much better surrogate marker for either prognostication or V-region status. ^{[35] [36] [37]} However, even though this assay is widely available, particularly in commercial reference laboratories, there can be significant variability in how the assay is performed, ^{[38] [39]} so that it is not always certain that a commercially available assay is the same as that in the published literature. For this reason, many researchers recommend caution in interpreting these results. Other attempts have been made to define prognostically significant subsets of patients with CLL using other combinations of markers,^[40] but these have not been adopted in routine practice.

The primary value of flow cytometry in chronic leukemias is its ability to demonstrate clonality in β-cell populations, based on restricted expression of one type of immunoglobulin light chain. ^{[21] [22] [23]}This feature is particularly valuable in the evaluation of patients with unexplained lymphocytosis ^{[41] [42]} and also is useful for staging patients with β-cell non-Hodgkin's lymphoma, ^{[43] [44]} because the presence of as little as 0.5% to 1% of a clonal β-cell population, or even less, can be demonstrated in the marrow or blood of some patients. The high sensitivity of flow cytometry for detecting clonal populations has, however, demonstrated small clonal β-cell populations in some patients without obvious lymphoma or leukemia.^[45] Thus, the finding of a small marrow clone, in the absence of other evidence of lymphoma, should be interpreted with caution, analogous to finding a monoclonal gammopathy in a patient without evidence of myeloma.

Some controversy exists about the applicability of flow cytometry to patients with myeloma. Whereas plasma cells have a characteristic phenotype, including very bright expression of CD38 and expression of CD138, ^{[46] [47]} plasma cells are underrepresented on marrow aspirates studied by flow compared with the prevalence of cells on films or in biopsies. Nevertheless, the unique phenotype of plasma cells makes them easy to recognize, and with the use of membrane-permeabilization techniques, it is easy to demonstrate cytoplasmic immunoglobulin (Ig) light-chain restriction.^[47] It is of more significance that neoplastic plasma cells usually have abnormal phenotypes. Although no single specific phenotypic abnormality permits distinction between benign monoclonal gammopathy and myeloma, the relative proportion of abnormal versus normal plasma cells has been suggested to be a predictor of behavior.^[48] Moreover, flow cytometric detection of circulating plasma cells or persistence of an abnormal phenotype after therapy is predictive of outcome in patients with myeloma. ^{[49] [50] [51] [52]}

Clonality of T cell populations also can be demonstrated by flow cytometry, although the method is more complex and is not as widely available as that used for B cells. This technique is based on demonstration of restriction of V-beta gene use in T-cell leukemias. ^{[53] [54]} T-cell malignancies also often show abnormal T-cell phenotypes, most often characterized by loss of a normal pan-T antigen, or expression of a T-cell antigen at abnormal intensity. ^{[55] [56]} Because certain small, unusual, and even clonal T-cell populations may be seen in small numbers in non-neoplastic conditions, this technique has more limited sensitivity than demonstration of a clonal β-cell population. However, when abnormal T cells account for more than a very small percentage of cells, multiparameter flow cytometry easily demonstrates them and plays a significant role in categorizing these uncommon tumors.

Residual Disease Detection in Chronic Lymphoproliferative Disorders

As with acute leukemia, MRD can be detected by flow cytometry in chronic lymphoproliferative disorders. The most work has been done with CLL. Although detection of light-chain-restricted clones is difficult when the level is much below 0.1%, aberrant phenotypes that allow detection with a sensitivity of at least 1 in 10^-4 can be detected in most cases of CLL. ^{[57] [58]} Just as in acute leukemia, the presence of MRD in patients considered to be in remission by standard criteria is associated with an adverse prognosis,^[58] whereas clearance of MRD after therapy is associated with a good outcome.^[59]

Solid Tumors: Analysis for DNA Content

Numerous fluorescent dyes bind stoichiometrically to cellular DNA and thus can provide an accurate assessment of DNA content of cells. In a single-parameter fluorescence histogram, tumors with abnormal numbers of chromosomes show a distinct peak separate from the normal G₀/G₁ peak of diploid cells. Such tumors are referred to as aneuploid. Moreover, cells progressing through the cell cycle show incrementally increased levels of DNA while they are in S phase, and those in G₂ or in mitosis show exactly twice the amount of DNA as that represented by the G₀/G₁ population. Thus, integration of the area under the curve between the G₀/G₁ and G₂/M peaks gives the proportion of cells in S phase, commonly referred to as S-phase fraction. A variety of software packages are available to calculate S-phase fraction from these histograms.

DNA content analysis in tumors was one of the earliest applications of flow cytometry in tumors. Many studies have attempted to define the prognostic significance of either ploidy or S-phase fraction in a large number of different solid tumors. This proliferation of studies was made possible by the finding that fixed, paraffin-embedded tissue sections could be used for DNA analysis by flow, which resulted in many retrospective studies on archival material for which outcome was already known.^[60] However, the literature is confusing and contradictory, and the early promise of this measurement as an important diagnostic and prognostic marker in cancer has not been realized. Although some studies have demonstrated prognostic significance to measurements of ploidy, and especially S-phase fraction in a number of tumors—most specifically bladder, prostate, and breast cancer—many studies conflict, with the result that this technology has not been widely embraced in clinical oncology. In some practices, it is used occasionally to help manage certain subgroups of patients with some cancers. For example, some practices use S-phase fraction to help manage patients with early-stage, node-negative breast cancer, whereas others use ploidy of superficial bladder cancers to help identify patients whose tumors might progress. One reason for the lack of acceptance of this measurement is the difficulties that have been encountered in standardization. S-phase fraction, in particular, has shown very poor interlaboratory reproducibility.^[61] The ultimate effect of these technical problems is that it is very difficult for an individual laboratory to offer clinicians a test result that helps them decide how to manage a particular patient. The inability of this technology to make significant inroads in the clinic is unfortunate, because some more recent studies, by using highly sophisticated analytic methods, suggest that in breast cancer, at least, both ploidy and S-phase fraction are powerful independent prognostic markers.^[62] A detailed summary of all the controversies is outside the scope of this chapter, but several reviews have been published. ^{[63] [64]}

FUTURE OF FLOW CYTOMETRY IN CLINICAL ONCOLOGY

Flow cytometry in the clinical laboratory is at something of a crossroads. On the one hand, improvements in standardization and the technology itself have made it possible to develop easy-to-operate instruments that fit well with the model of the clinical laboratory. On the other hand, with the notable exception of MRD assessment, no significant growth has occurred in the applicability of this technology to cancer. Empirical classification of leukemia and lymphoma with new markers has not, in general, been fruitful. At the same time, technologic advances in cancer diagnosis currently seem to be focusing on methods of assessing genetic lesions in cancer. However, although genetic abnormalities clearly produce cancer, they do so through production of abnormal proteins, and detection of a number of different proteins in specific cell populations is what flow cytometry does best.

Thus it would appear that flow cytometry is well suited to validate, and to translate into clinical practice, many of the exciting findings that derive from genomics. One of the areas that has seen significant attention is the flow cytometric detection of phosphorylated signaling molecules. These studies have greatly added to our understanding of the operation of signaling pathways,^[65] can be used to demonstrate alterations in signaling in leukemia, and are ideally suited to use as surrogate markers to validate the action of new drugs. ^{[66] [67]} While these measurements have not yet been incorporated into routine clinical practice, they are already being used in clinical trials,^[68] and it is likely that the most robust and informative of them will soon find their way into the routine laboratory.

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