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107 Adding space to TiME by sequential immunofluorescence: a new dimension for the spatial biomarkers painting in a clinical cohort of lymphoma case
  1. Pedro Machado Almeida1,
  2. Albertr Santamaria-Martínez2,
  3. Emilie Peres1,
  4. Elisa Oricchio2,
  5. Diego Dupouy1 and
  6. Marco Cassano1
  1. 1Lunaphore Technologies, Tolochenaz, Vaud, Switzerland
  2. 2École Polytechnique Fédérale de Lausanne, Lausanne, VD, Switzerland
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Hematological malignancies exhibit diverse phenotypes and origins, reflecting the wide range of immune cells they arise from. The pathological assessment of these cancers relies on multiple complementary analyses, including morphological evaluation and immunohistochemistry.1 The current gold standard for immunohistochemical assessment in most clinical pathology laboratories is single-plex, chromogenic immunohistochemistry. Thus, this approach requires a separate FFPE tissue section for each individual antibody, which prevents evaluating all markers on the same section, leading to potential misdiagnosis due to biological variabilities between sequential sections, increased workload, rapid tissue exhaustion, and further exposing the patients to the risks and complications of acquiring additional tissue and delays in diagnosis and treatment.2 3 Currently, flow cytometry-based immunophenotyping allows for the evaluation of multiple biomarkers but lacks spatial information due to the lack of tissue context. The recent surge of automated multi-omics platforms now offers an ideal toolkit to perform multiplex immunostaining on a single FFPE tissue section in an automated, reproducible manner.

Methods We used the COMET™ platform to perform sequential immunofluorescence (seqIF™) with a hyperplex immuno-oncology panel containing 34 markers on reactive lymphadenopathy and Non-Hodgkin Lymphoma (NHL) FFPE samples. The resulting OME-TIFF images containing DAPI for nuclear staining, two autofluorescence channels for background subtraction, and 34 single marker channels were used for downstream image analysis.4 Background subtraction, cell segmentation, and single-cell phenotyping were performed using the HORIZON™ software.5

Results Here, we successfully optimized and verified the hyperplex panel using a single reactive lymphoid hyperplasia case, demonstrating the feasibility of 34-plex staining with COMET™ aimed at identifying different cellular phenotypes within the tumor immune microenvironment (TiME). Subsequently, the hyperplex panel was intuitively transferred in a few days to a cohort of 7 patients with minimal tissue samples required, to further investigate the distribution of cellular phenotypes across multiple cases.

Conclusions Finally, we demonstrate that our automated workflow reduces the experimental turnover and sample consumption while enabling a comprehensive understanding of the tumor-stroma milieu in lymphoma. This proof-of-concept work provides compelling evidence for the technical feasibility of multiplexing biomarker detection from a single FFPE lymphoma section. The intuitive cohort expansion capability of COMET™ further enhances its value in understanding the TiME while minimizing experimental and sample requirements. The future integration of multiplexing technologies into clinical practice holds great potential to render the pathological assessment of hematological malignancies more accurate, robust, reproducible, and streamlined than the current gold standard.


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  4. Migliozzi D, et al. Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections Microsyst Nanoeng, 2019;6(5):59.

  5. Navikas V, et al. Automated multiplex immunofluorescence workflow to interrogate the cellular composition of the tumor microenvironment, Cancer Res, 2023;83(7 Supplement).

Ethics Approval Approved by the Commission cantonale d’éthique de la recherche sur l’être humain of the Canton de Vaud (CER-VD, project-ID 2020-02532)

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