RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance.

BACKGROUND: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. METHODS: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. RESULTS: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. CONCLUSIONS: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in cutaneous T cell lymphoma.

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors are novel targeted therapies being evaluated in clinical trials for cutaneous T-cell lymphoma (CTCL). However, data in regard to tumor biology are limited with these agents. In the present study we analyzed the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the proteasome inhibitor bortezomib on human CTCL cells. Four CTCL cell lines (SeAx, Hut-78, MyLa, and HH) were exposed to bortezomib and/ or SAHA at different concentrations. Cell viability was quantified using the MTT assay. In addition, apoptosis and generation of reactive oxygen species were analyzed. Both agents potently inhibited cell viability and induced apoptosis. After 48 h of incubation, IC50 of bortezomib was noted at 8.3 nm, 7.9 nm, 6.3 nm, and 22.5 nm in SeAx, Hut-78, HH, and MyLa cells, respectively. For SAHA, the IC50 values were at 0.6 microm in SeAx cells, 0.75 microm in Hut-78 cells, 0.9 microm in HH cells, and 4.4 microm in MyLa cells. Importantly, combined treatment resulted in synergistic cytotoxic effects, as indicated by Combination indices values <1 using the median effect method of Chou and Talalay. We furthermore found that combined treatment with both agents lead to a decreased proteasome activity, an upregulation of the cell regulators p21 and p27 and increased expression of phosphorylated p38. In addition, we showed that SAHA reduced the vascular endothelial growth factor production of CTCL cells. Our results demonstrate that bortezomib and SAHA synergistically induce apoptosis in CTCL cells and thus provide a rationale for clinical trials of combined proteasome and histone deacetylase inhibition in the treatment of CTCL.

Emerging drugs in cutaneous T cell lymphoma.

BACKGROUND: Mycosis fungoides (MF) represents the most common type of primary cutaneous T cell-lymphomas (CTCL), which are characterized by a clonally proliferation of malignant CD4+ lymphocytes in the skin. OBJECTIVE: Skin-directed treatment regimens, like phototherapy and corticosteroids, are commonly used in early stages; systemic treatments and chemotherapies are used in advanced stages. Because conventional treatments usually end in a transient remission without curative results, there is a high need for new therapeutic strategies with acceptable side effects. METHODS: Literature and reference research was done by using the data bank PubMed, and updates of ongoing studies were taken out of ASCO and ASH annual meeting abstracts. RESULTS/CONCLUSIONS: This article gives an overview of the various medications in current use, with emphasis on emerging drugs with novel therapeutic targets.