Pseudo-spikes are common in histologically benign lymphoid tissues

Abstract

T cell receptor gene rearrangement is a classic marker of T cell clonality and is a useful adjunct in the diagnosis of T cell lymphomas and leukemias. Rearranged V-J gene segments amplified by polymerase chain reaction (PCR) are traditionally analyzed by polyacrylamide gel electrophoresis. We and others have analyzed TCR-gamma PCR products using capillary gel electrophoresis, which produces single nucleotide resolution and provides improved diagnostic sensitivity over conventional methods. However, with this marked increase in resolution and sensitivity, it is necessary to re-define normal variation of TCR-gamma gene rearrangement in control tissues to allow appropriate interpretation of monoclonality if present. Using DNA capillary gel electrophoresis, we examined the spectrum of normal patterns for TCR-gamma in a variety of T-cell-rich, histologically benign tissue types, including spleen, lymph node, tonsil, and blood, and compared this with the patterns in T cell lymphoma samples. We defined relative peak heights as h1/h2, where h1 represents the peak height of the largest peak above the normally distributed population, and h2 represents the peak height of the normally distributed curve. We found spikes in almost 20% of histologically benign samples with relative peak heights that were more than 0.5 and up to 1.5. We designated these as pseudo-spikes, because they may be mistaken for monoclonal spikes. In contrast, the relative peak height of the T cell lymphoma samples that showed clonal rearrangement was much higher than that of the pseudo-spikes, being at least 2 in 11/11 and at least 3 in 10/11 cases. Our data suggest that peaks with relative height of at least 3 represent a true clonal population in diagnostic samples. Peaks with relative heights of less than 1.5 may be insignificant, while peaks with relative heights between 1.5 to 3 may warrant further evaluation. Although capillary gel electrophoresis is superior in assessing T cell clonality, caution must be exercised when interpreting results, because pseudo-spikes appear to be common in benign tissues with lymphoid populations and are not necessarily indicative of clonal malignant T cell population.

Figure 1.

Definition of relative peak heights. Relative peak heights were expressed as h₁/h₂, where h₁ represents the peak height of the largest peak above the normally distributed population, and h₂ represents the peak height of the normally distributed curve. A and B show h₁/h₂ to be the correctly expressed relative peak height even when the peak in question is displaced toward the edge of the normally distributed population of molecules (B). B illustrates an alternative ratio (h₁/h₃) where measurement of the peak height of the polyclonal curve was made at the position of the largest peak above the normally distributed population (h₃). This produces a falsely elevated ratio that may be misinterpreted as clonal. C and D illustrate how h₂ is measured in an actual sample where the polyclonal curve often has jagged edges.

Figure 2.

Chromatographs from benign lymphoid tissues. The y axis is relative fluorescent units and the x axis is size in bases. Size standards corresponding to 150, 160, and 200 bases are shown by arrowheads and the area of TCR-γ amplification is defined by the horizontal double-headed arrow. A: Tonsillitis specimen demonstrating noise. Spleen with capsular laceration and reactive follicular hyperplasia. (B), spleen with capsular hematoma. (C), and histologically normal spleen from patient with chronic pancreatitis and pseudocyst (D), demonstrating pseudo-spikes. Classic bell-shaped curve (E), skewed bell-shaped curve (F), and bell-shaped curve with sharp peak (G), demonstrating range of polyclonal electropherograms. H: Oligoclonal pattern in a normal spleen sample from a patient with adenocarcinoma of the pancreas.

Figure 3.

Chromatographs from T cell lymphoma samples demonstrating monoclonal spikes. A: Bone marrow with peripheral T cell lymphoma. B: Lymph node with peripheral T cell lymphoma. C: Bone marrow with peripheral T cell lymphoma. Symbols and axes as in Figure 2 .

Figure 4.

CGE versus PAGE in detecting small clonal populations. We mixed varying proportions of a monoclonal population (Jurkat cell line, J) into a heterogeneous polyclonal T cell population (N) and analyzed the mixes with PAGE (A) and CGE (B). Symbols and axes as in Figure 2 . In A, the fourth lane from the left shows TCR-γ amplification products from the Jurkat cell line, and the adjacent third lane shows TCR-γ and β-globin PCR products amplified in a single multiplexed reaction from the cell line. With CGE, the presence of the monoclonal population is definitively detected at 1:25 J:N dilution, whereas the presence of a monoclonal band could not be definitively ascertained on PAGE even at 1:10 J:N dilution.