A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment

Pulsawatdi, Amélie Viratham; Craig, Stephanie G.; Bingham, Victoria; McCombe, Kris; Humphries, Matthew P.; Senevirathne, Seedevi; Richman, Susan D.; Quirke, Philip; Campo, Leticia; Domingo, Enric; Maughan, Tim; James, Jacqueline; Salto-Tellez, Manuel

doi:10.1002/1878-0261.12764

Cited by 78 publications

(68 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, we also found mIF occasionally suffered an increase sensitivity of cell identification in the sample, the implication for DIA was therefore a detection of an increased number detected in the DAPI channel. This too may also lend more confirmation to the evidence that tissue thickness should be considered in any mIF study design, as we have noted in a previous report when considering the impact of autofluorescence [ 17 ]. While the comparative PD-L1 score was highly correlated between DAB and mIF, the increased sensitivity of the mIF technique may account for the lower number of overcalls in comparison to DIA in DAB IHC.…”

Section: Discussionsupporting

confidence: 79%

“…The high correlation and reproducibility of biomarker data generated between DAB and mIF is fundamental in the establishment of mIF as a robust method of biomarker interrogation. Several studies have reported their experience with this assessment, and while our data, and those reported previously by our group [ 17 ], indicate that single mIF staining is highly correlated with IHC, some provide only anecdotal evidence [ 19 ]. Koelzer et al reported comparable staining between mIF chromogenic IHC staining in their panel (PD-L1, CD3, CD8, CD57, and PD-1) [ 15 ].…”

Section: Discussionsupporting

confidence: 60%

“…Validation of the staining sequence was conducted to accurately control the specific conditions applied in the multiplex, as described [ 4 , 17 ]. This included the sequential ordering of primary antibodies to ensure representative epitope stability and therefore comparable expression of singleplex DAB staining vs. singleplex IF staining following several rounds of epitope retrieval.…”

Section: Methodsmentioning

confidence: 99%

“…Advancements in multiplexing technology bring the potential to deploy mIF in a clinical setting [ 4 , 14 , 15 ]. Studies are also beginning to show that applying DIA to mIF can significantly improve biomarker detection [ 11 , 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

Sidi

Bingham

Craig

et al. 2020

Cancers

Self Cite

View full text Add to dashboard Cite

Multiplex immunofluorescence (mIF) and digital image analysis (DIA) have transformed the ability to analyse multiple biomarkers. We aimed to validate a clinical workflow for quantifying PD-L1 in non-small cell lung cancer (NSCLC). NSCLC samples were stained with a validated mIF panel. Immunohistochemistry (IHC) was conducted and mIF slides were scanned on an Akoya Vectra Polaris. Scans underwent DIA using QuPath. Single channel immunofluorescence was concordant with single-plex IHC. DIA facilitated quantification of cell types expressing single or multiple phenotypic markers. Considerations for analysis included classifier accuracy, macrophage infiltration, spurious staining, threshold sensitivity by DIA, sensitivity of cell identification in the mIF. Alternative sequential detection of biomarkers by DIA potentially impacted final score. Strong concordance was observed between 3,3’-Diaminobenzidine (DAB) IHC slides and mIF slides (R2 = 0.7323). Comparatively, DIA on DAB IHC was seen to overestimate the PD-L1 score more frequently than on mIF slides. Overall, concordance between DIA on DAB IHC slides and mIF slides was 95%. DIA of mIF slides is rapid, highly comparable to DIA on DAB IHC slides, and enables comprehensive extraction of phenotypic data and specific microenvironmental detail intrinsic to the sample. Exploration of the clinical relevance of mIF in the context of immunotherapy treated cases is warranted.

show abstract

Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

Sidi

Bingham

Craig

et al. 2020

Cancers

Self Cite

View full text Add to dashboard Cite

show abstract

“…For multiplexing immunofluorescence, 4 micron sections were obtained from cases observed to demonstrate perinuclear STING by DAB IHC. Sections were stained with validated methods, as described 19,20 . Staining was performed on a Leica Bond-Max (Leica Biosystems UK, Milton Keynes), using Opal 4-Color Automation IHC Kit (CK/STING/DAPI) (Cat.…”

Section: Immunohistochemistrymentioning

confidence: 99%

The clinical and molecular significance associated with STING signaling in estrogen receptor-positive early breast cancer

Parkes

Humphries

Gilmore

et al. 2020

Preprint

View full text Add to dashboard Cite

STING signaling in cancer is a crucial component of response to immunotherapy and other anti-cancer treatments. Conversely, STING signaling can promote tumor invasion and metastasis. Currently, there is no robust method of measuring STING activation in cancer. Here, we describe an immunohistochemistry-based assay with digital pathology assessment of STING in tumor cells. Using this novel approach, we identify perinuclear-localized expression of STING (pnSTING) in estrogen receptor-positive (ER+) breast cancer as an independent predictor of good prognosis, associated with immune cell infiltration and upregulation of immune checkpoints. Tumors with low pnSTING are immunosuppressed with increased infiltration of “M2” -polarised macrophages. In ER-disease, pnSTING does not have a significant prognostic role, and STING appears to be uncoupled from interferon responses. Importantly, a gene signature defining low pnSTING expression in ER+ disease is predictive of poor prognosis in independent datasets. Low pnSTING is associated with chromosomal instability, MYC amplification and mTOR signaling, suggesting novel therapeutic approaches for this subgroup.

show abstract

The Porto European Cancer Research Summit 2021

et al. 2021

View full text Add to dashboard Cite

Key stakeholders from the cancer research continuum met in May 2021 at the European Cancer Research Summit in Porto to discuss priorities and specific action points required for the successful implementation of the European Cancer Mission and Europe's Beating Cancer Plan (EBCP). Speakers presented a unified view about the need to establish high‐quality, networked infrastructures to decrease cancer incidence, increase the cure rate, improve patient's survival and quality of life, and deal with research and care inequalities across the European Union (EU). These infrastructures, featuring Comprehensive Cancer Centres (CCCs) as key components, will integrate care, prevention and research across the entire cancer continuum to support the development of personalized/precision cancer medicine in Europe. The three pillars of the recommended European infrastructures – namely translational research, clinical/prevention trials and outcomes research – were pondered at length. Speakers addressing the future needs of translational research focused on the prospects of multiomics assisted preclinical research, progress in Molecular and Digital Pathology, immunotherapy, liquid biopsy and science data. The clinical/prevention trial session presented the requirements for next‐generation, multicentric trials entailing unified strategies for patient stratification, imaging, and biospecimen acquisition and storage. The third session highlighted the need for establishing outcomes research infrastructures to cover primary prevention, early detection, clinical effectiveness of innovations, health‐related quality‐of‐life assessment, survivorship research and health economics. An important outcome of the Summit was the presentation of the Porto Declaration, which called for a collective and committed action throughout Europe to develop the cancer research infrastructures indispensable for fostering innovation and decreasing inequalities within and between member states. Moreover, the Summit guidelines will assist decision making in the context of a unique EU‐wide cancer initiative that, if expertly implemented, will decrease the cancer death toll and improve the quality of life of those confronted with cancer, and this is carried out at an affordable cost.

show abstract

A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment

Cited by 78 publications

References 32 publications

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

PD-L1 Multiplex and Quantitative Image Analysis for Molecular Diagnostics

The clinical and molecular significance associated with STING signaling in estrogen receptor-positive early breast cancer

The Porto European Cancer Research Summit 2021

Contact Info

Product

Resources

About