Abstract
BackgroundAnaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples.MethodsA novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in.ResultsThe 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051).ConclusionThe newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions.
Highlights
Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed
Rearrangement of the anaplastic lymphoma kinase (ALK) gene is an oncogenic driver event typically occurring in young non-smokers suffering from a KRAS/EGFR wild-type lung adenocarcinoma [1, 2]
We have developed an ALK immunofluorescence protocol for the microfluidic tissue processor (MTP) device to be performed on formalin-fixed paraffin-embedded (FFPE) cancer tissue section
Summary
Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. Rearrangement of the anaplastic lymphoma kinase (ALK) gene is an oncogenic driver event typically occurring in young non-smokers suffering from a KRAS/EGFR wild-type lung adenocarcinoma [1, 2]. The ALK status is routinely assessed in diagnostic surgical pathology by either or a combination of the four methods. Despite the initial poor performances of ALK on lung adenocarcinoma, immunohistochemistry is currently considered reliable and cheap, due to employment of signal amplification system and optimized anti-ALK antibodies, such as clone 5A4 (Novocastra) or D5F3 (Ventana), with the latter being approved by FDA (Food and Drug Administration) as companion diagnostic test for ALK rearrangements [6]
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