Molecular techniques used in the diagnosis of cutaneous lymphoma

Cutaneous lymphomas are a heterogenic group of conditions often difficult to diagnose. The diagnosis requires careful correlation between clinical presentation pathology and molecular analysis. Molecular analysis includes inmunophenotyping, clonality assays and rarely chromosomal analysis. The importance of molecular analysis hinges on two main reasons: firstly to confirm the diagnosis and secondly to further characterize the nature of the lymphoma. In addition, molecular analysis may provide some further insight on the origin of the malignancy, for example if it is primarily cutaneous or if the skin is a secondary site of involvement.

by Dr Belén Rubio González and Dr Joan Guitart

Recently, cancer has been defined by unlimited growth of cells derived from a single mutated cell or a clonality expansion. The detection of a monoclonal population may help to distinguish a lymphoma from a reactive process. However, on the one hand, clonality by itself does not imply malignancy and, on the other hand, a negative clonality result does not rule out a malignant condition. During this process, genes encoding the antigen receptor immunoglobulin (Ig) for B cells and the T-cell receptor (TCR) for T cells are rearranged as commonly seen in primed lymphocytes, resulting in a wide diversity of unique antigen receptors providing high antigenic specificity.

The clonal nature of several skin conditions may help us recognize pre-malignant stages or the concept of cutaneous lymphoid dyscrasias (CLD) which has been recently introduced and includes parapsoriasis, pigmented purpuric dermatosis, idiopathic follicular mucinosis, pityriasis lichenoides, syringolymphoid hyperplasia with alopecia, and idiopathic generalized erythroderma (pre-Sézary). Although almost all of these conditions never progress to a frank malignancy, they have the potential risk of converting into cutaneous T-cell lymphoma (CTCL). The recognition of a T-cell clone may identify these dermatoses, which have been difficult to categorize in the past.

Clonality methods
T-cell clonality studies are based on the detection of specific T-cell receptor gene rearrangements (TCR-GR) by Southern blot analysis (SBA) or polymerase chain reaction (PCR). We should expect that the tumour cells contain identical TCR-GRs, reflecting a monoclonal T-cell population.

SBA used to be the gold standard for detection of T-cell clonality, but the procedure is laborious and lengthy. Furthermore, fresh or frozen tissue and radioactive probes are required. If this method is used, the clonal population must represent at least 5% of the total DNA extracted, which includes cells other than T-cells decreasing the sensitivity of the test. For the reasons above, SBA has been gradually replaced by PCR techniques.

The overall sensitivity of PCR-based methods for detection of T cell clonality ranges between 70 and 90%, with specificity range depending on the sample population. The test amplifies extracted DNA using primers directed against the TCR beta, gamma and delta chains. The gamma chain gene is most commonly used because of the lower complexity of the gene. Adding probes to the beta TCR gene allows for a higher sensitivity and specificity of the clonal analysis.

In the case of B-cells, PCR uses primers for four conserved reliable targeted regions for immunoglobulin heavy-chain. In our experience the sensitivity of PCR for the immunoglobulin heavy chain is lower than for T-cell clonality. The detection of light chain restriction by immunophenotypic test (often referred as monotypical immunoglobulin expression) is also consistent with a clonal B-cell population. This can be accomplished with immunohistochemistry at the protein level or in situ hybridization at the RNA level. Monoclonality can also be demonstrated with flow cytometry targeting kappa and lambda light chain expression at the B-cell membrane. An international consensus on B- and T-cell clonality assays was established with the BIOMED-1 proposal.

In most of the conventional PCR methods monoclonality is defined by the presence of a band after high-resolution capillary gel electrophoresis of the PCR product. Using temperature- or chemical-gradient gel electrophoresis can enhance separation of DNA products. After that, fluorescent fragment analysis using consensus primers for the TCR gene and the fluorescence input is analysed by capillaroscopy. Furthermore, clonal definition should be confirmed using multiple PCR probes labelled with different fluorochromes.

The detection of a dominant T-cell clone, defined as the same PCR product at different sites (two skin biopsies, skin and blood, skin and lymph node, etc.) implies dissemination of a prevailing T-cell clone, and has been associated with a higher incidence of tumour progression. Clonal heterogeneity has been reported in patients with early stage or indolent mycosis fungoides (MF) and in CLD conditions without a malignant process.

The value of the detection of circulating clonal T-cells in peripheral blood has been debated. That is much more common in patients with erythrodermic MF (42%) compared to other lower stages (12.5%). It may also help in distinguishing a dominant CTCL clone from innocent cytotoxic T-cell clones, which are often detected in the blood of elderly patients.

In the context of palpable lymphadenopathy, detection of the same clone in the lymph node and the skin CTCL lesions may indicate a poor prognosis, similar to the identification of lymphoma by histology.

T-cell clonality and significance
TCR clonality should be tested for in skin and blood samples at the time of diagnosis when a cutaneous lymphoma is suspected. The detection of a dominant clone in both sites is important to confirm the diagnosis and for prognostic guidance. T-cell clonality is particularly helpful in the early stage of an MF which does not include sufficient clinical or microscopic evidence for the diagnosis. TCR gamma clonality was positive in 53% of the patch stage and in 100% of plaque or tumour stage in different series. An increased rate of clonality was observed in connection with more advanced cutaneous disease and higher histopathological diagnostic score.

False-positive monoclonal and oligoclonal bands may be identified in inflammatory dermatosis, where the T-cell infiltrate is sparse. Amplification of TCR-GRs from a few T-cells may result in a false-positive clone or ‘pseudomonoclonality’. A pseudoclone is infrequently associated with a malignant T-cell process. Repeating the analysis using the same DNA template or fresh DNA extraction may solve the problem because in reactive conditions, the predominant PCR product typically varies in repeated analysis of the same sample. In contrast, in lymphomas, dominant TCR clones are reproducible and should be routinely verified to confirm monoclonality.

A correlation between TCR clonality by PCR methods and response to treatment has been suggested in several studies. The absence of a detectable clone in CTCL was associated with a higher rate of complete remission, but was not necessarily associated with improved survival.

Also immunophenotypic and immunogenotypic assays have been used to monitor the response of CTCL to therapy. The concept of minimal residual disease is defined as the persistence of the tumour T-cell clone in tissue or blood despite clinical complete remission status. Minimal residual disease as detected by deep sequencing methods may help identify patients at risk of relapse but the real prognosis is still uncertain. In the future, the presence or absence of the dominant or persistent clone may guide our therapeutic approach, aiming for more durable remissions while minimizing the adverse effects of therapy.

Other methods used in the olecular diagnosis of cutaneous lymphomas
Flow cytometry analysis
Blood flow cytometry analysis (FCA) is routinely performed in erythrodermic patients to rule out Sézary syndrome (SS). This method is based on the abnormal expression of various surface markers of malignant T-cells compared with normal T-cells. Other helpful findings are the demonstration of overwhelming dominance of specific T-cell subsets (clusters of differentiation CD4 vs CD8) and the loss of one or more pan-T-cell antigens (i.e. CD2, CD3, CD5, and CD7). A high CD4 : CD8 ratio of more than 10 : 1 and loss of CD7 and CD26 are the most reliable findings in SS. However, low CD7 expression has lower specificity because some inflammatory diseases also show the same deletion. The addition of CD26 to standard T-cell panels enhances the sensitivity of FCA in the diagnosis of SS.

Moreover, flow cytometry is able to detect a clonal population by using antibodies against different subsets of T-lymphocytes based on the expression of V beta family antibodies. This is used mainly as a research tool because the extensive panel of antibodies is expensive, incomplete and does not include the entire spectrum of V beta families.

Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) involves annealing of fluorescently labelled nucleic acid probes with complementary DNA or RNA sequences and the subsequent detection of these probes within fixed cells. FISH is used to detect major chromosomal gains or losses, as well as specific translocations, and requires a target specific probe. Although FISH is not routinely used in the diagnosis of cutaneous lymphomas, recent publications have shown its potential for future applications in various areas.

Genomic analysis by microarray assays or comparative genomic hybridization
Comparative genomic hybridization (CGH) allows the identification of chromosomal imbalances but it is not able to identify specific genes involved due to its measurement resolution. The microarray-based CGH is more precise, and chromosomal imbalances can be quantified and defined appositionally. A high frequency of gains in chromosomes 1, 7, 8, and 17 and losses of chromosomes 5, 9, and 13 was demonstrated using array-based CGH for identification of genomic differences between SS and MF.

Conclusion
Molecular diagnosis, in combination with a meaningful correlation with histological results and clinical presentations can provide an important tool in the evaluation of cutaneous lymphoid infiltrate. While PCR-based clonality techniques need to be interpreted with caution, modern capillaroscopy methods offer clone-specific data that allow us to improve the accuracy for diagnosis, prognosis and staging implication.

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The authors
Belén Rubio González* MD and Joan Guitart MD
Northwestern Medical Hospital, Chicago, IL, USA
*Corresponding author
E-mail: rubiogonzalezbelen@gmail.com