The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force

Metastatic sites.

<p>Metastatic sites.</p

PGK1 and CXCR4 expression, proliferation and inhibition of CXCR4 of neuroblastoma cell lines.

<p>Kelly (<b>A</b>) and SH-EP Tet-21/N (<b>B</b>) neuroblastoma cells were immunostained for PGK1 and CXCR4 expression (<b>Immunohistochemistry</b>). Both cell lines show a positivity for CXCR4 and react to treatment with 20 µg AMD3100 with an inhibition of proliferation (<b>MTT-assay</b>), although only SH-EP Tet-21/N cells reach a significant level of growth reduction. On examination of PGK1 protein expression levels (<b>Western blot</b>) after 48 h of CXCR4 receptor inhibition, treatment with 20 µg AMD3100 leads to a downregulation of PGK1 protein (45 kDa) in SH-EP Tet-21/N but not in Kelly cells. Tubulin (55 kDa) served as control.</p

Overall Survival.

<p>For Kaplan-Meier survival analysis patients were grouped according to positive and negative PGK1 expression. All patients that died during the follow-up period showed positive PGK1 expression, while none of the PGK1 negative patients died. Overall survival of neuroblastoma patients with a PGK1 negative expression was significantly better than that of PGK1 positive patients (<i>p = 0.003</i>).</p

Patient characteristics.

<p>Patient characteristics.</p

FISH analysis in ESCC patients.

<p>Green signals represent the <i>FGFR1</i> gene while red signals correspond to the centromere of chromosome 8. (A) high-level amplification of <i>FGFR1</i> showing 10–20 gene signals and 2–4 centromere signals with a ratio of 6.16. (B) Heterogeneous amplification of <i>FGFR1</i> as indicated by presence of two distinct cancer areas with FGFR1 amplification and without FGFR1 amplification. These two areas are separated by the dotted line. (C) Normal <i>FGFR1</i> gene and centromere 8 signals.</p

FGFR1 expression.

<p>FGFR1 expression.</p