Advances in Cutaneous Molecular Medicine Supplement Clinical Expertise

• The 454 Pyrosequencing (Roche). This powerful platform, one of several next generation sequencing technologies that allows ultra deep sequencing of entire exons, was used to resolve mutation discrepancies between the cobas 4800 BRAF V600 test and the Sanger sequencing method during the vemurafenib trials, the authors noted. It has a mean error rate of only 1.07%, with more than half of the errors at sites of homopolymers, has the lengths of individual reads of DNA sequences of more than 500 base pairs, and can be performed in less than a day.

• Cobas 4800 BRAF V600 Mutation Test. The cobas 4800 BRAF V600 Mutation Test is based on the principles of allele-specific real time PCR, and targets a predefined 116-base pair sequence of the BRAF gene on exon 15. The device, which is intended to identify those with BRAF V600E who might benefit from therapy with vemurafenib, has a sensitivity for detecting BRAF V600E on FFPE samples of melanoma of more than 99%, and a specificity of 88%. The sensitivity appears comparable with the other platforms, including Sequenom and HRM.

Although the platform is not designed to screen for nonrecurrent genetic mutations in melanoma, BRAF V600E appears to account for the highest percentage of mutations in cutaneous melanomas, the authors said.

Cutaneous Lymphoma Analysis

In cutaneous lymphomas, molecular analysis serves to confirm the diagnosis in cases in which the clinical and/or pathologic presentations do not provide a diagnosis and to further characterize the nature of the lymphoma, according to Dr. Janyana M.D. Deonizio and Dr. Joan Guitart.

While the gold standard for diagnosis is a solid clinicopathologic correlation, molecular analysis provides for a more concrete diagnosis that helps both the patient in facing the diagnosis, and the clinician in proceeding with the most appropriate treatment plan (Sem. Cut. Med. Surg. 2012;31:234-40).

Specifically, through immunophenotyping and clonality analysis, molecular analysis helps discern whether the lymphoma is primarily cutaneous or systemic with secondary skin involvement, and it subclassifies the tumor.

Methods for establishing T-cell clonality include Southern blot analysis (SBA) and PCR for detection of specific T-cell receptor gene arrangements (TCR-GR). SBA used to be the gold standard, but has been gradually replaced by PCR techniques which are less laborious and lengthy. PCR sensitivity for T-cell clonality detection ranges from 70% to 90%.

"Ideally, TCR clonality should be checked at the time of diagnosis in skin and blood. ... The detection of a dominant clone is important not only to confirm diagnosis but also for some prognostic guidance," they wrote, explaining that T-cell cloning is particularly helpful when early-stage mycosis fungoides is being considered in the differential diagnosis.

It does have limitations, however. False-positive monoclonal or oligoclonal bands may be identified in inflammatory dermatoses when T-cell infiltrates are sparse, resulting in "pseudomonoclonality," which is infrequently associated with a malignant T-cell process, they noted.

"Repeating the analysis using the same DNA template or fresh DNA extraction may solve the problem because in reactive conditions, the predominant PCR products typically vary in repeated PCR analyses of the same sample. In contrast, in neoplastic T-cell proliferations, dominant TCR clones are reproducible and should be routinely verified to confirm monoclonality," they noted.

Some studies suggest a correlation between TCR clonality by PCR and response to treatment; the absence of a detectable clone in cutaneous T-cell lymphoma (CTCL) has been associated with a higher rate of complete remission – although not necessarily with improved survival.

Immunophenotypic and immunogenotypic assays have been used to monitor the response of CTCL to therapy, define remission, and detect early relapse, thereby improving assessment of disease activity.

Flow cytometry analysis, for example, is "an efficient and sensitive method to detect and enumerate abnormal cells in the peripheral blood or any other cell suspension," they wrote. It can also be performed on leukocyte suspension from skin biopsies, and it provides prognostic information.

"Lower counts of circulating CD8+ lymphocytes and higher white cell counts in CTCL patients are associated with a less favorable prognosis," the authors noted.

FISH is used to detect major chromosomal gains or losses and specific translocations using a target-specific probe. Although FISH is not routinely used in the diagnosis of cutaneous lymphomas, it does appear to have potential future applications in various areas, according to recent publications.

Finally, genomic analysis by microarray-based comparative genomic hybridization is allowing quantification and appositional defining of chromosomal imbalances. While still confined to the research arena, this technology is providing some insight into the molecular pathogenesis of CTCL, the reported.

The advances in molecular diagnostics that are outlined in this series of articles are not limited to skin cancers and tumors. Additional articles, for example, addressed the role of genetic and molecular analysis in alopecia and in genodermatoses. Together they underscore the need for, and substantiate the ability of the specialty to take on leadership roles in molecular medicine, noted Dr. Gerami, of the department of dermatology at Northwestern University in Chicago.