Abstract
Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.
Highlights
With the advancement of personalized cancer medicine, precise molecular profiling of tumors is gaining significant importance in routine diagnostic pathology[1,2]
Continuous signal quantification of double IF staining using microfluidic precision immunofluorescence. For this proof-of-concept study, we used the Microfluidic Tissue Processor” (MTP) to perform IF staining assays on formalin-fixed paraffin-embedded (FFPE) sections of surgically resected human invasive breast carcinoma samples (Fig. 1A), retrieved from the archives of the Institute of Pathology at the University Hospital of Lausanne (Switzerland)
Microfluidic precision IF proved to be very powerful in terms of quantifying Ag expression in histopathological tissue sections, significantly increasing the precision of the information that can be obtained by an immunoassay
Summary
With the advancement of personalized cancer medicine, precise molecular profiling of tumors is gaining significant importance in routine diagnostic pathology[1,2]. If a routine method that precisely quantifies Ag expression in tissues while preserving the morphology could be established, would this reduce the requirement for expensive complementary gene analysis and increase the precision of diagnosis, prognosis and the success of targeted therapies, in clinical trials and routine patient care In this context, as clinical pathology moves from qualitative to quantitative, immunofluorescence (IF) is gaining relevance in the research settings and laboratory-developed tests, mainly due to its increased capacity to measure the signal intensity of one or more biomarkers as compared to traditional chromogenic techniques[7,10]. We selected 25 invasive breast carcinoma cases whose HER2 biomarker expression ranged across a large range of values known by routine diagnostics, as assessed by the two conventional methods used (i.e.: IHC and FISH), and for which several cases presented equivocal results Using these cases, we show that, continuous quantification of HER2 protein expression by microfluidic precision immunofluorescence, one can estimate the number of copies of the coding gene, as assessed by FISH. We anticipate that our method has the potential to provide automated and precise continuous quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required for personalized cancer therapy
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