Treatment of mycosis fungoides with brentuximab vedotin: Assessing CD30 expression by immunohistochemistry and quantitative real‐time polymerase chain reaction

Abstract

Brentuximab vedotin (BV), an anti-CD30 antibody conjugated with the cytotoxic agent monomethyl auristatin E (MMAE), has achieved significant therapeutic success in CD30-positive cutaneous T-cell lymphomas (CTCL). 1 Microscopic evaluation of immunohistochemically stained slides of tumoral tissue is regarded as the gold standard to select appropriate patients for CD30-targeted therapy. 2 Among the different subgroups of CTCL, there is high variability of CD30 expression. 3 Surprisingly, in mycosis fungoides (MF), which is the most common CTCL, profound treatment response to BV has also been observed when CD30 expression, as assessed by immunohistochemistry, was quite negligible (<5%, ie, expression levels that are commonly stated by pathologists as being negative). 4,5 This hitherto incompletely understood phenomenon-which might be related to insufficient sensitivity of immunohistochemical CD30 detection-is, however, of immediate clinical relevance: based on the approval conditions of BV, such "CD30-negative" patients might not be considered eligible for treatment with BV: that is, these patients would not receive one of the most efficient treatment options currently available for advanced CTCL.Motivated by these observations, we aimed to evaluate whether the detection of CD30 mRNA expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis would be more sensitive than immunohistochemistry when assessing the CD30 expression status of tumoral tissue.A total of 18 samples (cryo-preserved, and formalin-fixed, paraffin-embedded tissue) obtained from skin lesions of seven patients with MF being treated with BV were analyzed (Table 1). The study was approved by the ethical committee of the University of Würzburg, and all patients provided informed consent. Biopsy samples were obtained before, during, and after BV treatment. The percentage of CD30 expression levels as assessed by immunohistochemistry using the anti-CD30 antibody Ber-H2 (Dako, Agilent Technologies, Santa Clara, California) was categorized into four different groups based on the frequency of CD30-positive cells (%) (Score 0, <5%; Score 1, 5%-25%; Score 2, 26%-50%; Score 3, 51%-75%; Score 4, >75%) (Figure 1A,B). In addition, staining intensity was assessed (faint/medium/high). qRT-PCR of mRNA isolated from corresponding cryo-preserved tissue sections was performed using TaqMan assay. For CD30 mRNA detection, the following specific CD30 primers and Taqman probe were used: CD30_fw: GAA TTC GGC AGA AGC TCC AC; CD30_rv: CTC CTC CTG GGT CTG GAA TC; CD30_probe: [6FAM]CCG GTC CAG ACC TCC CAG CC [BHQ1]. ACTB expression served as endogenous control (ß-actin_fw: GCG AGA AGA TGA CCC AGA TC; ß-actin_rv: CCA GTG GTA CGG CCA GAG G; ß-actin_probe: [6FAM]CCA GCC ATG TAC GTT GCT ATC CAG GC[BHQ1]). The relative CD30 mRNA expression was calculated by the ΔΔC t method (Sample #5 served as calibrator) and was compared with the immunohistochemical data (Figure 1B). CD30 expression at the mRNA and protein level-as investigated by the two differe...