Opportunities for Hyperspectral and Multispectral Imaging

BackgroundIn patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration.MethodsWe treated 43 patients with predominantly microsatellite stable RAS/BRAF wild-type mCRC on a phase II trial combining chemotherapy with the epidermal growth factor receptor antibody cetuximab and the programmed cell death ligand 1 (PD-L1) antibody avelumab. We performed next-generation gene panel sequencing for mutational typing of tumors and liquid biopsy monitoring as well as digital droplet PCR to confirm individual mutations. Translational analyses included tissue immunohistochemistry, multispectral imaging and repertoire sequencing of tumor-infiltrating T cells. Detected PD-L1 mutations were mechanistically validated in CRISPR/Cas9-generated cell models using qRT-PCR, immunoblotting, flow cytometry, complement-dependent cytotoxicity assay, antibody-dependent cytotoxicity by natural killer cell degranulation assay and LDH release assay as well as live cell imaging of T cell mediated tumor cell killing.ResultsCirculating tumor DNA showed rapid clearance in the majority of patients mirroring a high rate of early tumor shrinkage. In 3 of 13 patients expressing the high-affinity Fcγ receptor 3a (FcγR3a), tumor subclones with PD-L1 mutations were selected that led to loss of tumor PD-L1 by nonsense-mediated RNA decay in PD-L1 K162fs and protein degradation in PD-L1 L88S. As a consequence, avelumab binding and antibody-dependent cytotoxicity were impaired, while T cell killing of these variant clones was increased. Interestingly, PD-L1 mutant subclones showed slow selection dynamics reversing on avelumab withdrawal and patients with such subclones had above-average treatment benefit. This suggested that the PD-L1 mutations mediated resistance to direct antitumor effects of avelumab, while at the same time loss of PD-L1 reduced biological fitness by enhanced T cell killing limiting subclonal expansion.ConclusionThe addition of avelumab to standard treatment appeared feasible and safe. PD-L1 mutations mediate subclonal immune escape to avelumab in some patients with mCRC expressing high-affinity FcγR3a, which may be a subset experiencing most selective pressure. Future trials evaluating the addition of avelumab to standard treatment in MSS mCRC are warranted especially in this patient subpopulation.Trial registration numberNCT03174405.

Download Full-text

Multispectral Imaging for Automated Fish Quality Grading

2020 IEEE 15th International Conference on Industrial and Information Systems (ICIIS) ◽

10.1109/iciis51140.2020.9342726 ◽

2020 ◽

Author(s):

Dhananjaya Jayasundara ◽

Lakshitha Ramanayake ◽

Neranjan Senarath ◽

Sanjaya Herath ◽

Roshan Godaliyadda ◽

...

Keyword(s):

Multispectral Imaging ◽

Fish Quality ◽

Quality Grading

Download Full-text

747 Evaluation of immune microenvironment of primary lung cancer and synchronous liver metastasis with multispectral imaging system

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0747 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A795-A795

Author(s):

Hyeonbin Cho ◽

Jae-Hwan Kim ◽

Ji-Hyun Kim

Keyword(s):

T Cells ◽

Liver Metastasis ◽

Multispectral Imaging ◽

Immune Cell ◽

Imaging System ◽

Cytotoxic T Cells ◽

Primary Lung Cancer ◽

Helper T Cells ◽

Synchronous Liver Metastasis ◽

Immune Microenvironment

BackgroundCancer immunotherapy (CIT) has substantially improved the survival of cancer patients. However, according to recent studies, liver metastasis was reported to predict worse outcomes for CIT. The main objective of the study is to evaluate the differences in the immune microenvironment (IME) between the primary lung cancer (PL) and synchronous liver metastasis (LM) using a multispectral imaging system.MethodsSix immune markers (CD4, CD8, CTLA-4, granzyme B (GZB), Foxp3 and PD-L1) were analyzed using a multiplex IHC system and inForm program (Akoya) on paired lung-liver samples of 10 patients. Cells were categorized into tumor nest and stroma, and cell counts per unit area were measured for comparison.ResultsThe number of tumor-infiltrating cytotoxic T cells (TIL) in PL (262.5 cells/mm2) was higher than that of LM (113.3 cells/mm2). Additionally, the ratio between the number of TIL and non-TIL was greater in PL (0.31) compared to that of LM (0.26). A similar trend appeared for Helper T cells and regulatory T cells (Treg), as PL consisted of higher numbers of T cells (791.8 Helper T cells/mm2, 195.7 Treg/mm2) than LM (626.3 Helper T cells/mm2, 121.3 Treg/mm2). However, cytotoxic T cells exhibiting GZB+ and CTLA-4- were fewer in PL (140.2 cells/mm2) than in LM (203.3 cells/mm2), and the ratio is 0.69. The mean number of GZB+ TIL in PL (32.5 cells/mm2) was lower than in LM (35.3 cells/mm2), and their proportions among total TIL counts were 0.12 and 0.31, respectively. In PL, GZB+: GZB- ratio is 0.16 while the ratio is 1.91 for LM. A fewer number of TILs exhibiting GZB suggests that PL has lower efficiency in immune response than LM. Another crucial checkpoint receptor that inhibits immune response, CTLA-4, was more prevalent in PL, with CTLA-4+: CTLA-4- ratio in Treg being 0.36 in PL, compared to 0.11 in LM. The tumor proportion score (TPS) of PD-L1 was higher in PL than LM (40.0 vs. 6.6).ConclusionsIn our study, we showed the differences in the numbers of TIL or regulatory T cells and expressions of immune checkpoint receptors (PD-L1, CTLA-4), which significantly influence outcomes for CIT. The study is ongoing to confirm different IME between the PL and LM groups in a larger tumor cohort.ReferencesPeng, Jianhong, et al., Immune Cell Infiltration in the Microenvironment of Liver Oligometastasis from Colorectal Cancer: Intratumoural CD8/CD3 Ratio Is a Valuable Prognostic Index for Patients Undergoing Liver Metastasectomy. Cancers 2019 Dec; 11(12): 1922. https://doi.org/10.3390/cancers11121922Tumeh, Paul C., et al., Liver Metastasis and treatment outcome with Anti-PD-1 monoclonal antibody in patients with melanoma and NSCLC. Cancer Immunol Res 2017 May; 5(5): 417–424. doi: 10.1158/2326-6066.CIR-16-0325Parra, E.R., Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis Approaches; Streckfus, C.F., Ed.; IntechOpen: London, UK, 2018; pp. 1–13. doi: 10.5772/intechopen.80380Ribas, A., Hu-Lieskovan, S., What does PD-L1 positive or negative mean?. The Journal of Experimental Medicine 2016;213(13):2835–2840. https://doi.org/10.1084/jem.20161462

Download Full-text

Automated Detection of Bleeding in Capsule Endoscopy using on-chip Multispectral Imaging Sensors

IEEE Sensors Journal ◽

10.1109/jsen.2020.3034831 ◽

2020 ◽

pp. 1-1

Author(s):

Mohammad Reza Mohebbian ◽

Md Hanif Ali Sohag ◽

Seyed Shahim Vedaei ◽

Khan A. Wahid

Keyword(s):

Capsule Endoscopy ◽

Multispectral Imaging ◽

Automated Detection ◽

On Chip ◽

Imaging Sensors

Download Full-text

Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002197 ◽

2021 ◽

Vol 9 (7) ◽

pp. e002197

Author(s):

Janis M Taube ◽

Kristin Roman ◽

Elizabeth L Engle ◽

Chichung Wang ◽

Carmen Ballesteros-Merino ◽

...

Keyword(s):

Breast Carcinoma ◽

Multispectral Imaging ◽

Immune Cell ◽

Clinical Laboratory ◽

Predictive Biomarkers ◽

Spatial Arrangement ◽

Tissue Microarrays ◽

Fluorescent Detection ◽

Nsclc Tissue ◽

Tissue Sections

BackgroundEmerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments.MethodsA six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs.ResultsComparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75).ConclusionsAssay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.

Download Full-text

Optimized Method for Mapping Inorganic Pigments by Means of Multispectral Imaging Combined with Hyperspectral Spectroscopy for the Study of Vincenzo Pasqualoni’s Wall Painting at the Basilica of S. Nicola in Carcere in Rome

Minerals ◽

10.3390/min11080839 ◽

2021 ◽

Vol 11 (8) ◽

pp. 839

Author(s):

Lucilla Pronti ◽

Giuseppe Capobianco ◽

Margherita Vendittelli ◽

Anna Candida Felici ◽

Silvia Serranti ◽

...

Keyword(s):

Multispectral Imaging ◽

Imaging System ◽

Principal Component ◽

Reflectance Spectroscopy ◽

Wall Painting ◽

High Spectral Resolution ◽

Spectral Difference ◽

Color Palette ◽

A Minor ◽

Nir Reflectance Spectroscopy

Multispectral imaging is a preliminary screening technique for the study of paintings. Although it permits the identification of several mineral pigments by their spectral behavior, it is considered less performing concerning hyperspectral imaging, since a limited number of wavelengths are selected. In this work, we propose an optimized method to map the distribution of the mineral pigments used by Vincenzo Pasqualoni for his wall painting placed at the Basilica of S. Nicola in Carcere in Rome, combining UV/VIS/NIR reflectance spectroscopy and multispectral imaging. The first method (UV/VIS/NIR reflectance spectroscopy) allowed us to characterize pigment layers with a high spectral resolution; the second method (UV/VIS/NIR multispectral imaging) permitted the evaluation of the pigment distribution by utilizing a restricted number of wavelengths. Combining the results obtained from both devices was possible to obtain a distribution map of a pictorial layer with a high accuracy level of pigment recognition. The method involved the joint use of point-by-point hyperspectral spectroscopy and Principal Component Analysis (PCA) to identify the pigments in the color palette and evaluate the possibility to discriminate all the pigments recognized, using a minor number of wavelengths acquired through the multispectral imaging system. Finally, the distribution and the spectral difference of the different pigments recognized in the multispectral images, (in this case: red ochre, yellow ochre, orpiment, cobalt blue-based pigments, ultramarine and chrome green) were shown through PCA false-color images.

Download Full-text

Altered Spatial Composition of the Immune Cell Repertoire in Association to CD34+ Blasts in Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemia

Cancers ◽

10.3390/cancers13020186 ◽

2021 ◽

Vol 13 (2) ◽

pp. 186

Author(s):

Marcus Bauer ◽

Christoforos Vaxevanis ◽

Haifa Kathrin Al-Ali ◽

Nadja Jaekel ◽

Christin Le Hoa Naumann ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndromes ◽

Myeloid Leukemia ◽

Multispectral Imaging ◽

Immune Cell ◽

Genetic Alterations ◽

Spatial Proximity ◽

Secondary Acute Myeloid Leukemia ◽

Acute Myeloid

Background: Myelodysplastic syndromes (MDS) are caused by a stem cell failure and often include a dysfunction of the immune system. However, the relationship between spatial immune cell distribution within the bone marrow (BM), in relation to genetic features and the course of disease has not been analyzed in detail. Methods: Histotopography of immune cell subpopulations and their spatial distribution to CD34+ hematopoietic cells was determined by multispectral imaging (MSI) in 147 BM biopsies (BMB) from patients with MDS, secondary acute myeloid leukemia (sAML), and controls. Results: In MDS and sAML samples, a high inter-tumoral immune cell heterogeneity in spatial proximity to CD34+ blasts was found that was independent of genetic alterations, but correlated to blast counts. In controls, no CD8+ and FOXP3+ T cells and only single MUM1p+ B/plasma cells were detected in an area of ≤10 μm to CD34+ HSPC. Conclusions: CD8+ and FOXP3+ T cells are regularly seen in the 10 μm area around CD34+ blasts in MDS/sAML regardless of the course of the disease but lack in the surrounding of CD34+ HSPC in control samples. In addition, the frequencies of immune cell subsets in MDS and sAML BMB differ when compared to control BMB providing novel insights in immune deregulation.

Download Full-text

Zinc Seed Priming Improves Spinach Germination at Low Temperature

Agriculture ◽

10.3390/agriculture11030271 ◽

2021 ◽

Vol 11 (3) ◽

pp. 271

Author(s):

Muhammad Imran ◽

Asim Mahmood ◽

Günter Neumann ◽

Birte Boelt

Keyword(s):

Low Temperature ◽

Temperature Stress ◽

Multispectral Imaging ◽

Germination Rate ◽

Cost Effective ◽

Seed Priming ◽

Seedling Development ◽

Low Temperature Stress ◽

Early Seedling ◽

Early Seedling Development

Low temperature during germination hinders germination speed and early seedling development. Zn seed priming is a useful and cost-effective tool to improve germination rate and resistance to low temperature stress during germination and early seedling development. Spinach was tested to improve germination and seedling development with Zn seed priming under low temperature stress conditions. Zn priming increased seed Zn concentration up to 48 times. The multispectral imaging technique with VideometerLab was used as a non-destructive method to differentiate unprimed, water- and Zn-primed spinach seeds successfully. Localization of Zn in the seeds was studied using the 1,5-diphenyl thiocarbazone (DTZ) dying technique. Active translocation of primed Zn in the roots of young seedlings was detected with laser confocal microscopy. Zn priming of spinach seeds at 6 mM Zn showed a significant increase in germination rate and total germination under low temperature at 8 °C.

Download Full-text