Chronic lymphocytic leukemia other diagnostic studies

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]; Associate Editor(s)-in-Chief: Raviteja Guddeti, M.B.B.S. [2]

Other Diagnostic Tests

Biopsy

Bone marrow biopsy is not required in all cases of CLL. It is used to establish the findings of anemia and thrombocytopenia. It can distinguish between thrombocytopenia of peripheral destruction (in the spleen) and that due to marrow infiltration.

Determining clonality

The diagnosis of CLL is based on the demonstration of an abnormal population of B lymphocytes in the blood, bone marrow, or tissues that display an unusual but characteristic pattern of molecules on the cell surface. This atypical molecular pattern includes the co-expression of cells surface markers cluster of differentiation 5 (CD5) and cluster of differentiation 23 (CD23). In addition, all the CLL cells within one individual are functionally inert and clonal, that is genetically identical. In practice, this is inferred by the detection of only one of the mutually exclusive antibody light chains, kappa or lambda, on the entire population of the abnormal B cells. Normal B lymphocytes consist of a stew of different antibody producing cells resulting in a mixture of both kappa and lambda expressing cells. The lack of the normal distribution of kappa and lambda producing B cells is one basis for demonstrating clonality, the key element for establishing a diagnosis of any B cell malignancy (B cell Non-Hodgkin lymphoma).

Flow Cytometry

Clonality is confirmed by the combination of the microscopic examination of the peripheral blood and analysis of the lymphocytes by flow cytometry. The latter is easily accomplished on a small amount of blood. A flow cytometer is an instrument that can examine the marker molecule expression on individual cells in fluids. This is accomplished using antibodies with fluorescent tags recognized by the instrument. In CLL, the lymphocytes are genetically clonal, of the B cell lineage (express marker molecules CD19 and CD20), and characteristically express the marker molecules CD5 and CD23.

Fluorescence in situ hybridization (FISH)

In addition to the maturational state, the prognosis of patients with CLL is dependent on the genetic changes within the neoplastic cell population. These genetic changes can be identified by fluorescent probes to chromosomal parts using a technique referred to as fluorescent in situ hybridization (FISH). Compared with fluorescence in-situ hybridization (FISH), conventional metaphase cytogenetics play ONLY a MINOR prognostic role in CLL, so far, due to technical problems resulting from a limited proliferation of CLL cells in-vitro. Therefore conventional cytogenetics may define subgroups with a high risk of progression. FISH can be done (in CLL) on dividing and non-dividing cells. FISH doesn't tell about IgVH mutations nor does it define the presence of trisomy either. FISH is useful as long as there are CLL cells to test; you can't do it in a complete response (CR).The application of FISH to study interphase nuclei gives important prognostic information with B-cell CLL, especially for patients with 11q-, trisomy 12, 13q- and 17q-. The procedure of FISH involves cell cultures which are prepared after metaphase and prometaphase chromosomes are fixed to a glass slide. A DNA probe is then hybridized onto the chromosome; the probe is labeled with fluorochrome which can be detected with fluorescent microscopy. FISH can be done on dividing and non-dividing cells. Inversions will be missed as probes detect sequences not precise locations. Small mutations, such as small deletions and insertions, will also be missed. FISH is a cytogenetic technology that looks at 200-500 blood cells (obtained with a bone marrow biopsy). Because of the small size it is not as sensitive as PCR. (PCR has extreme sensitivity as well as being quite specific). PCR amplifies a fragment of DNA. It is at least 2-3 logs more sensitive than cytogenetic technology like FISH. PCR measurement requires a sample blood draw which is less invasive and intense than a bone marrow biopsy (with FISH). Four main genetic aberrations are recognized in CLL cells that have a major impact on disease behavior.

1. Deletions of part of the short arm of chromosome 17 (del 17p13) which target the cell cycle regulating protein p53 (a tumore suppressor gene) are particularly deleterious. Patients with this abnormality have significantly short interval before they require therapy and a shorter survival. This abnormality is found in 5-10% of patients with CLL.
2. Deletions of the long arm on chromosome 11 (del 11q22-q23) are also unfavorable although not to the degree seen with del 17p. The abnormality targets the ATM gene and occurs infrequently in CLL (5-10%).
3. Trisomy 12, an additional chromosome 12, is a relatively frequent finding occurring in 20-25% of patients and imparts an intermediate prognosis. It has a higher frequency of DNA aneuploidy.
4. Deletion of the long arm of chromosome 13 (del 13q14) is the most common abnormality in CLL with roughly 50% of patients with cells containing this defect. These patients (along with those of normal karyotype) have the best prognosis and most will live many years, even decades, without the need for therapy. The gene targeted by this deletion is a segment that likely produces small inhibitory RNA molecules that affect expression of important death inhibiting genes.

The presence of 17p- typifies cells that are resistant to fludarabine, alkylators and rituxumab. 11q- portends a decreased RR to fludrabine as well as an early relapse after bone marrow transplant (BMT). Both the 17p- and the 11q- have a decreased progression-free survival (PFS) and overall survival (OS).

Array-based karyotyping

Array-based karyotyping is a cost-effective alternative to FISH for detecting chromosomal abnormalities in CLL. Several clinical validation studies have shown >95% concordance with the standard CLL FISH panel.

References

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