Diagnosis of each of the hematologic malignancies requires a tissue biopsy or examination of a peripheral blood smear. In leukemias, the diagnosis typically can be made by examination of a peripheral blood smear and/or bone marrow biopsy and aspirate. For plasma cell neoplasms, the diagnosis is usually based on a bone marrow biopsy, whereas lymphomas are most commonly diagnosed by biopsy of a lymph node or another soft tissue site.
At the time of the initial biopsy, it is usually unknown whether a patient has a malignancy or a benign disorder leading to hyperplasia of myeloid or lymphoid cells, such as an infection or an inflammatory condition. The key to distinguishing between benign and malignant processes is clonality. Neoplasms originate from a single transformed cell and are therefore monoclonal. In contrast, reactive processes stem from the response of many different cells and are polyclonal.
Sometimes all that is required to make the distinction between benign and neoplastic processes is a glance at the tissue under the microscope; for example, replacement of normal tissue by a monomorphous population of abnormal cells is a hallmark of certain neoplasms (Fig. 19-1A). However, the make-up of other hematologic tumors is quite heterogeneous, mimicking the appearance of an exuberant reactive process (Fig. 19-1B). In such instances, molecular tests of clonality (described in a following section) play a critical role in establishing the diagnosis of a hematologic malignancy.
Varied morphology of hematologic malignancies. A) Acute lymphoblastic leukemia (bone marrow aspirate) is an example of a relatively monomorphic tumor composed of a population of lymphoblasts that are arrested at early stages of B-cell differentiation. B) Chronic myelogenous leukemia (CML, bone marrow biopsy), in contrast, is a tumor derived from hematopoietic stem cells in which maturation is preserved. As a result, marrow involved by CML is replaced by a heterogeneous tumor cell population that mimics the appearance of hyperplastic non-malignant bone marrow. As is typical of CML, most of the cells in this field consist of granulocytes at various phases of differentiation, including neutrophils, eosinophils, and several dark-staining basophils (b).
The mainstay of diagnosis remains the microscopic examination of tumor cells in smears or tissue sections, but morphology alone is insufficient to accurately diagnose and classify most hematologic malignancies. Thus, tumor samples are routinely examined by using several of the following complementary approaches:
Morphology. The appearance of tumor cells usually allows a diagnostician (typically a pathologist with training in hematopathology) to generate a short list of diagnostic possibilities. For example, the presence of blasts containing Auer rods, abnormal needle-like azurophilic granules (Fig. 19-2A), is diagnostic of an acute myeloid leukemia and would trigger the performance of other tests needed to further classify this type of tumor. Similarly, replacement of the marrow by an abnormal population of plasma cells would strongly support the diagnosis of multiple myeloma (Fig. 19-2B). However, it is often impossible to determine the lineage of a hematologic malignancy with certainty by morphology. For example, many B-cell and T-cell lymphomas cannot be reliably distinguished by appearance alone, and (as mentioned earlier) it may not even be possible to tell a reactive hyperplasia from a malignancy without additional tests.
Immunophenotyping refers to the staining of tumor cells with antibodies specific for antigens (mostly proteins) that are useful in the classification of hematologic malignancies. Some of the antigens that are particularly useful are summarized in Table 19-2. When fresh, unfixed tumor cells are available, antigens are often detected by flow cytometry (described in Chapter 1). In flow cytometry, cells are identified and enumerated based on their light scattering properties and their expression of various antigens, which are detected with antibodies tagged with fluorescent dyes. Flow cytometry is widely used in the diagnosis of acute leukemias and non-Hodgkin lymphomas. It is often also used to establish the clonality of mature B-cell tumors (Fig. 19-3). Mature B cells express only one type of immunoglobulin (Ig), which is composed of two heavy chains paired with either kappa or lambda light chains. In polyclonal B-cell proliferations, the ratio of kappa- to lambda-expressing cells is usually close to 1:1. However, because malignancies are derived from single transformed cells, in mature B-cell tumors, all of the cells express only kappa or lambda light chain. Due to its ability to quantify multiple markers on single cells, flow cytometry also can be used to detect cells expressing unusual combinations of antigens (another hallmark of certain hematologic malignancies), even within large populations of normal cells. Its chief limitation is that it must be done on fresh, unfixed cells, which are available only if a hematologic malignancy is suspected at the time the biopsy is obtained. A second method that avoids this limitation is immunohistochemistry (Fig. 19-4). Here, sections of tissues fixed in formalin and embedded in paraffin (the standard method of handling tissues in pathology departments) are placed on slides and incubated with specific antibodies. Staining is then developed by incubation with secondary antibodies linked to an enzyme such as horseradish peroxidase and a substrate that is converted by the enzyme to an insoluble colored precipitate. This method is widely used in the diagnosis of hematologic malignancies, particularly non-Hodgkin and Hodgkin lymphomas.
Histochemistry is used mainly in the diagnosis of acute leukemias. It is performed by incubating smears of marrow or blood with chemicals that differentially stain bone marrow cells of various lineages. Some stains involve the use of dyes that change color in the presence of specific leukocyte enzymes. Examples include stains for myeloperoxidase and specific esterase (both expressed by myeloblasts and more mature granulocytes) and nonspecific esterase (expressed by monoblasts and monocytes). Other stains react with non-enzymatic cellular constituents. The periodic acid–Schiff stain is used to detect glycogen, which is frequently present in the lymphoblasts of acute lymphoblastic leukemias and occasionally found in erythroblasts in myeloid neoplasms. The Prussian blue stain is used to detect non-heme iron, which accumulates in the mitochondria of red cell progenitors in certain myelodysplastic syndromes.
Cytogenetics. Tumor-specific cytogenetic aberrations can serve to establish the clonality of myeloid or lymphoid proliferations and in some instances are so characteristic of particular hematologic malignancies that they are required for the diagnosis. For example, all cases of chronic myelogenous leukemia are associated with chromosomal rearrangements that juxtapose the 3' portion of the ABL gene (normally on chromosome 9) with the 5' portion of the BCR gene on chromosome 22, thereby creating a chimeric BCR-ABL fusion gene with oncogenic activity. In most instances, this event occurs by way of a reciprocal translocation between chromosomes 9 and 22 that causes the formation of two abnormal chromosomes, one derived from chromosome 9 and the other from chromosome 22. The derivative chromosome 22 that bears the BCR-ABL fusion gene is often referred to as the Philadelphia chromosome (named for the city of its discovery). When fresh samples of marrow or blood are available, the Philadelphia chromosome usually can be directly visualized by determining the karyotype of tumor cells harvested in metaphase, the time in mitosis when chromosomes condense and can be recognized by their appearance (Fig. 19-5A). However, sometimes metaphases are not obtained, or complex rearrangements involving chromosomes 9 and 22 are present. In such instances, the BCR-ABL fusion gene and its reciprocal partner, an ABL-BCR fusion gene, can be detected directly by fluorescence in situ hybridization (FISH), in which DNA probes spanning the BCR and ABL loci are hybridized to interphase nuclei from tumor cells. In the example shown (Fig. 19-5B), the reciprocal rearrangement of BCR and ABL causes two abnormal fusion signals to appear, one due to the presence of the BCR-ABL gene on chromosome 22 and the second due to the nonfunctional ABL-BCR fusion gene on chromosome 9.
Molecular genetics. Sequence analysis of DNA or RNA isolated from tumor cells is playing an increasing part in the diagnosis of hematologic malignancies. For example, the diagnosis of polycythemia vera, a type of myeloproliferative disorder, requires the detection of activating mutations in the gene encoding the tyrosine kinase JAK2. Another common use of molecular -genetics is the assessment of clonality in lymphoid proliferations. Rearrangement and assembly of the immunoglobulin genes in B cells and the T-cell receptor genes in T cells create unique DNA sequences that are specific for that cell and all its progeny. Polyclonal, reactive lymphoid proliferations are composed of cells with many different sets of rearranged antigen receptor genes, whereas in lymphoid neoplasms all tumor cells share the same clonal rearrangements. Polymerase chain reaction (PCR)-based tests that involve the amplification of rearranged antigen receptor genes can -distinguish between polyclonal and monoclonal lymphoid proliferations with a high degree of sensitivity and specificity and are now in wide clinical use (Fig. 19-6). PCR analysis of reverse-transcribed messenger RNA (mRNA) from chronic myelogenous leukemia cells offers a reliable and sensitive means of detecting the BCR-ABL transcript.
TABLE 19-2Commonly Used Markers in the Diagnosis of Hematologic Malignancies ||Download (.pdf) TABLE 19-2 Commonly Used Markers in the Diagnosis of Hematologic Malignancies
|Markers ||Cells |
|CD34 ||Hematopoietic stem cells, early myeloid and lymphoid progenitors |
|CD117 (c-KIT) ||Early myeloid progenitors |
|CD13, CD33 ||Early and late myeloid forms |
|Terminal deoxyribonucleotidyl transferase ||Lymphoid progenitors |
|CD10, CD19 ||Early B-cell progenitors |
|CD20, surface immunoglobulin ||Mature B cells |
|CD10, BCL6 ||Follicular B cells |
|Cytoplasmic immunoglobulin ||Plasma cells |
|CD3, CD4, CD8 ||T-cell progenitors and mature T cells |
|CD5 ||T cells, certain B-cell tumors |
|CD15, CD30 ||Reed-Sternberg cells |
Examples of characteristic morphologies in hematologic malignancies. A) Auer rods (bone marrow aspirate). A myeloblast is shown that contains a single Auer rod, a needle-like, cytoplasmic, azurophilic granule that is pathognomonic of myeloid malignancies, most notably acute myeloid leukemia. B) Multiple myeloma (bone marrow biopsy). The marrow is largely replaced by a sheet of plasma cells with abnormally prominent, centrally placed nucleoli.
Use of flow cytometry to evaluate B-cell clonality. Cell suspensions prepared from two lymph node biopsy specimens obtained from patients suspected of having lymphoma were "gated" based on forward and side scatter (left hand panel), and the cells in the lymphocyte gate were then further characterized based on staining with antibodies specific for CD19 (tagged with the fluorescent dye APC), kappa immunoglobulin light chain (tagged with fluorescent dye FITC), and lambda immunoglobulin light chain (tagged with the dye PE). In A) roughly equal numbers of CD19-positive B cells expressing kappa and lambda light chain are present, a result consistent with the presence of a reactive polyclonal B-cell population. In B) there is a predominant population of cells expressing lambda light chain, indicating the presence of a monoclonal B-cell proliferation. Additional features of this population, including the expression of other markers and the morphology of the cells, would then be used to arrive at a specific diagnosis.
Use of immunohistochemistry to detect lineage-specific antigens. A diffuse large B-cell lymphoma is shown that has been stained with antibodies specific for CD20 (A), a surface protein that is expressed only by B lymphocytes, or CD3 (B), a marker that is restricted to T lymphocytes. The staining reaction is developed with a chromogenic substrate that produces a brown color; the nuclei of the cells have been counterstained with the blue dye hematoxylin. The tumor cells are positive for CD20, whereas a few reactive intermixed T cells are positive for CD3.
Cytogenetic analysis of hematologic malignancies. A) Karyotype, chronic myelogenous leukemia. Metaphase chromosomes stained with Giemsa dye are shown. The arrows denote two derivative chromosomes, a derivative 9 (der 9) and a derivative 22 (der 22), produced by a balanced translocation involving the BCR gene on chromosome 22 and the ABL gene on chromosome 9. The small der 22 chromosome, also known as the Philadelphia chromosome, contains the BCR-ABL fusion oncogene. B) Detection of a BCR-ABL fusion gene by fluorescence in situ hybridization (FISH). Interphase nuclei have been hybridized to fluorescently labeled probes specific for ABL and BCR. Cells containing a balanced (9;22) translocation have chimeric BCR-ABL and ABL-BCR genes that produce two fusion signals as well as one normal copy of ABL and one normal copy of BCR, each of which produce a normal signal.
Molecular detection of clonality by using antigen receptor gene rearrangements. Consensus oligonucleotide DNA primers that are complementary to TCRgamma V and J regions were used to perform the polymerase chain reaction (PCR) on DNA prepared from three different skin biopsy specimens suspected of being involved by cutaneous T-cell lymphoma, two of which were obtained from the same patient. The PCR products were then subjected to capillary electrophoresis, which can separate DNA fragments differing in size by as little as a single base pair. The sample from patient B produced multiple amplification products of differing sizes, a pattern that is consistent with a polyclonal reactive process. Differences in the sizes of the various V and J region segments in the TCRgamma gene produce a series of peaks and valleys in the normal sample. The individual products within each peak result from the random removal of bases by exonuclease and addition of bases by the enzyme terminal deoxyribonuclease at the time of joining of Vgamma and Jgamma segments in early T cells. In contrast, the two skin biopsy specimens from patient A produced two identical major PCR products, a result consistent with involvement of both sites by a single T-cell tumor that has rearranged both of its TCRgamma genes. (With permission from Dr. Janina Longtine.)
In addition to these tissue-based tests, other clinical and laboratory test findings weigh in on the diagnosis of certain hematologic malignancies. For example, tests that detect and quantify monoclonal antibodies or fragments of antibodies within the serum and the urine are important adjuncts in the diagnosis of plasma cell neoplasms, and radiologic studies to identify destructive bone lesions are part of the work-up of patients suspected of having multiple myeloma. The diagnosis of T-cell lymphoma is based in part on the pattern of tissue involvement, because specific subtypes of these diseases preferentially involve the skin, adipose tissue, gut, or spleen. It is believed that this behavior recapitulates the ability of normal T lymphocytes to home to specific tissues.