Molecular diagnosis in non-small-cell lung cancer: expert opinion on ALK and ROS1 testing

Data de publicació electrònica: 19-04-2021The effectiveness of targeted therapies with tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC) depends on the accurate determination of the genomic status of the tumour. For this reason, molecular analyses to detect genetic rearrangements in some genes (ie, ALK, ROS1, RET and NTRK) have become standard in patients with advanced disease. Since immunohistochemistry is easier to implement and interpret, it is normally used as the screening procedure, while fluorescence in situ hybridisation (FISH) is used to confirm the rearrangement and decide on ambiguous immunostainings. Although FISH is considered the most sensitive method for the detection of ALK and ROS1 rearrangements, the interpretation of results requires detailed guidelines. In this review, we discuss the various technologies available to evaluate ALK and ROS1 genomic rearrangements using these techniques. Other techniques such as real-time PCR and next-generation sequencing have been developed recently to evaluate ALK and ROS1 gene rearrangements, but some limitations prevent their full implementation in the clinical setting. Similarly, liquid biopsies have the potential to change the treatment of patients with advanced lung cancer, but further research is required before this technology can be applied in routine clinical practice. We discuss the technical requirements of laboratories in the light of quality assurance programmes. Finally, we review the recent updates made to the guidelines for the determination of molecular biomarkers in patients with NSCLC

Spindle cell squamous carcinoma of the tongue in a child

AbstractBackgroundSpindle cell carcinoma (SpCC) is an infrequent and aggressive type of squamous cell carcinoma (SCC) characterized by the proliferation of epithelial and mesenchymal components. Oral SCC in children is an extremely rare entity and the SpCC variant has been reported in one case in the paediatric patient literature.MethodsIn this paper, we report a case of SpCC of the tongue in an 11-year-old boy treated by on-block surgical resection and microvascular tissue reconstruction.ResultsAfter 14 months the patient is free of disease with easily intelligible speech and normal swallowing.ConclusionsDiagnosis and treatment of this rare tumour in this age group is a challenge because of the overlapping of histopathological features and the complex reconstruction required to achieve adequate aesthetic and functional results

Inflammatory cytokines and survival factors from serum modulate tweak-induced apoptosis in PC-3 prostate cancer cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted

TWEAK induces apoptosis in serum depleted androgen-independent PC-3 cells but not in androgen-sensitive LNCaP cells.

<p>Cell death was assessed by flow cytometry of DNA content. Hypodiploid cells were considered apoptotic. A) PC-3 cells, cultured with or without 10% FBS, were stimulated with TWEAK (100 ng/mL) alone or in presence of TNFα (30 ng/mL)/IFNγ (30 U/mL) for 24 hours. Mean (±SD) of four independent experiments. *p<0.002 vs control; #p<0.001 vs TWEAK alone; †p<0.002 vs 0% FBS with stimuli. B) LNCaP cells, cultured without FBS, were stimulated with TWEAK alone or in combination with TNFα/IFNγ for 24 hours. Mean (±SD) of three independent experiments. C) TWEAK, alone or in combination with TNFα/IFNγ, induces apoptosis in PC-3 cells in a dose-dependent manner. Mean (±SD) of three independent experiments. *p<0.002 vs control; #p<0.0001 vs control. D) Time-course of TWEAK-induced apoptosis in PC-3 cells, alone or in combination with TNFα/IFNγ. Mean (±SD) of three independent experiments. *p<0.002 vs control; #p<0.0001 vs control.</p

TWEAK induces cytochrome C release from the mitochondria in PC-3 cells.

<p>PC-3 cells treated with for 24 hours showed mitochondrial cytochrome C release. Confocal microscopy. Cytochrome C in red and DAPI in blue. (Magnification x400, detail x1000). Pictures representatives of three experiments.</p

TWEAK-induced PC-3 cell death has features of apoptosis. A)

<p>Flow cytometry of DNA content. Note hypodiploid, apoptotic cells (|――|) among those treated with TWEAK, or with TWEAK/TNFα/IFNγ for 24 hrs. Mean (±SD) of four experiments.*p<0.05 vs control. <b>B)</b> Characteristic shrunk, pyknotic, fragmented nuclei are present among DAPI-stained, TWEAK or TWEAK/TNFα/IFNγ-treated cells (arrows), but are infrequent among control or TNFα/IFNγ-treated cells. Magnification x400, detail x1000. <b>C)</b> PC-3 cells were cultured with or without serum and treated with TWEAK or TWEAK/TNFα/IFNγ for 24 hours. Cells were staining with Annexin V/PI and analyzed by flow cytometry. TWEAK increases both early (AnnexinV⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptosis under serum deprivation conditions. Graphic shows percentage of early and late apoptosis [(AnnexinV⁺/PI⁻)+(AnnexinV⁺/PI⁺)]. Mean (±SD) of three independent experiments. *p<0.008 vs control; #p<0.001 vs 0% FBS with stimuli.</p

PC-3 and LNCaP cells respond to TWEAK treatment.

<p>PC3 and LNCaP cells were stimulated with 100 ng/mL TWEAK for the indicated points, and Fn14 and MCP-1 mRNA levels were measured by RT-PCR. Both cell lines respond to TWEAK, but, the PC-3 response is more sustained compared to LNCaP cells. Mean (±SEM) of three independent experiments.*p<0.05 vs control.</p

TWEAK or Fn14 antagonism prevents apoptosis induced by TWEAK or TWEAK/TNFα/IFNγ in PC-3 cells.

<p>Cell death was assessed by flow cytometry of DNA content. <b>A)</b> PC-3 cells were pre-treated with anti-TWEAK neutralizing antibody (10 µg/ml), ITEM2 anti-Fn14 antibody (10 µg/ml), or anti-TNFα neutralizing antibody (10 µg/ml), added 1 hour before TWEAK. Cell death was assessed at 24 hours. Mean (±SD) of three independent experiments. *p<0.02 vs control; #p<0.0001 vs TWEAK alone. <b>B), C)</b> PC-3 cells were pre-treated with anti-TWEAK neutralizing antibody <b>(B)</b> or with ITEM2 anti-Fn14 antibody <b>(C)</b>, added 1 hour before TWEAK/TNFα/IFNγ. Cell death was assessed at 24 hours. Mean (±SD) of three independent experiments. *p<0.02 vs control; #p<0.0001 vs TWEAK/TNFα/IFNγ alone.</p

Fn14 expression in PC-3 cells is dependent on the microenvironment. A, B)

<p>PC-3 prostate cancer cells were cultured with 0% or 5% FBS for 3 or 24 hours. Fn14 mRNA expression was measured by quantitative RT-PCR <b>(A)</b> and Fn14 protein expression was studied by Western blot <b>(B)</b>. Mean (±SEM) of four independent experiments. *p<0.03 versus 0% FBS 3h; #p<0.003 vs 0% FBS 24 hours. <b>C, D)</b> Analysis of Fn14 mRNA expression <b>(C)</b>, and Fn14 protein expression <b>(D)</b> in PC-3 cells treated with TNFα (30 ng/mL)/IFNγ (30 u/mL) for 3 or 24 hours. Mean (±SEM.) of four independent experiments. #p<0.03 vs control 24 hours. <b>E)</b> Representative Western blot of Fn14 in PC-3 cells cultured with 0%FBS, 5%FBS or TNFα/IFNγ for 3 and 24 hours.</p