Tumour Infiltrating Lymphocytes in Follicular Lymphoma - additional data

Published: 4 January 2021| Version 3 | DOI: 10.17632/274xbhc5rx.3
Anna Maria Tsakiroglou,
Sue Astley,
Manàs Dave,
Martin Fergie,
Elaine Harkness,
Adeline Rosenberg,
Matthew Sperrin,
Catharine West,
Richard Byers,
Kim Linton


The composition of the tumour microenvironment in follicular lymphoma (FL) is a relevant factor in determining disease progression and treatment response. This dataset is a collection of 349 FL diagnostic tissue micro-array (TMA) cores from 130 patients, stained using multi-plex immunofluorescence for: • CD4+ cells • Cytotoxic T cells (CD8+) • T regulatory cells (Tregs [CD4+FOXP3+]) • Macrophages (CD68+) • PD1+ lymphocytes • B cells/follicular dendritic cells (CD21+) • DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain Changes from previous version 2 ---------- - Raw .im3 image files provided - Spectral library images provided - Labels of nuclear annotations in 16bit format Cohort -------- FL patients according to the WHO 2008 classification were identified from the archives of The Christie NHS Foundation Trust, Manchester, UK. The study was conducted with approval by the North-West Multi-centre Ethics Committee (03/08/016) and according to the Declaration of Helsinki. Examination of the records of 350 FL patients in a random order identified 262 patients meeting the inclusion criteria: adult patients with previously untreated FL; diagnosed from incisional or excisional biopsy; and treated at first presentation with radiotherapy, watchful waiting or a combination of chemotherapy and rituximab immunotherapy. Pre-treatment biopsies were requested for 262 patients, of which 130 had sufficient tissue for analysis. A histological diagnosis of FL was confirmed by an expert haemato-pathologist (R.B). Note: Three patients (FL_59, FL_106 and FL_129) have grade 3b, which is considered equivalent to Diffuse Large B-cell lymphoma. Image format and software compatibility --------------------------------------------- Each image represents a single TMA core as a .im3 raw multispectral image format, containing the following spectra: • DAPI (nuclear) • 650 fluorophore signal for CD68 marker (membrane) • 570 fluorophore signal for CD21 marker (membrane) • 540 fluorophore signal for CD8 marker (membrane) • 690 fluorophore signal for PD1 marker (membrane) • 620 fluorophore signal for CD4 marker (membrane) • 520 fluorophore signal for FOXP3 (nuclear) • Auto-fluorescence The commercial software inForm 2.4 (Akoya Biosciences) is compatible with this format. The images can be unmixed with inForm software by using the spectral library provided. Annotations ------------- Annotations are provided for nuclear segmentation. In a set of 41 small patches the outlines have been drawn manually for 69780 nuclei. A patch from the DAPI channel and corresponding label are given as .tif images. Data structure ---------------- The data is split across multiple different datasets, all referenced below. They include: • FL_0-129 zip files: Raw images for 130 patients, each zip file a single patient. • nuclear_segmentation_annotations.zip: The nuclear segmentation annotations. • spectral library.zip: The raw images used to build the spectral library with inForm software.


Steps to reproduce

Immunofluorescence method --------------------------------- Regions of interest were identified by a pathologist (R.B) and cores were extracted in triplicate from the formalin-fixed, paraffin embedded (FFPE) blocks to construct five TMA blocks. The multi-plex immunofluorescence protocol is publicly available in dx.doi.org/10.17504/protocols.io.49ygz7w. Briefly, 4μm tissue sections underwent 6 repeated cycles of indirect immunofluorescence with tyramide signal amplification using the Ultra Discovery (Roche, Basel, Switzerland) auto-stainer. The OpalTM fluorophores (520, 540, 570, 620, 650, 690 [Akoya Biosciences, Menlo Park, CA, USA]) were used for signal detection. After each cycle the heat retrieval step that followed stripped the primary and secondary antibodies, while the fluorophores remained on the epitopes. DAPI was used as a counterstain to observe nucleated cells. Scanning and spectral unmixing ------------------------------------ Sections were scanned with a Vectra 3.5 microscope (Akoya Biosciences) multispectrally at 20x magnification (0.49μm/ pixel). Phenochart 1.0.8 software (Akoya Biosciences) was used to recognise the TMA grid and enable scanning each core individually. After manual inspection some cores were excluded because of poor quality and artefacts, and 349 cores remained. Linear spectral unmixing to separate the 6 fluorophore and DAPI spectra was carried out in inForm 2.4 software (Akoya Biosciences). For this purpose a spectral library containing the individual fluorophore spectra was pre-built using single-plex assays of positive control samples. An unstained control to obtain the auto-fluorescence spectrum is also included.


The University of Manchester, Christie NHS Foundation Trust, Cancer Research UK Manchester Institute


Image Segmentation, Lymphocyte, Macrophage, Dendritic Cell, Immunohistochemistry, Histopathology, Microenvironment, Multiplexing, Cytotoxic T-Cell, T-Helper Cell, Regulatory T Cell, Follicular Lymphoma