Immune infiltrate diversity confers a good prognosis in follicular lymphoma

From Springer Nature via Jisc Publications RouterHistory: received 2020-08-25, accepted 2021-04-14, registration 2021-04-15, pub-electronic 2021-04-30, online 2021-04-30, pub-print 2021-12Publication status: PublishedFunder: Manchester Biomedical Research Centre; doi: http://dx.doi.org/10.13039/100014653; Grant(s): IS-BRC-1215–20007Funder: Manchester Cancer Research CentreAbstract: Background: Follicular lymphoma (FL) prognosis is influenced by the composition of the tumour microenvironment. We tested an automated approach to quantitatively assess the phenotypic and spatial immune infiltrate diversity as a prognostic biomarker for FL patients. Methods: Diagnostic biopsies were collected from 127 FL patients initially treated with rituximab-based therapy (52%), radiotherapy (28%), or active surveillance (20%). Tissue microarrays were constructed and stained using multiplex immunofluorescence (CD4, CD8, FOXP3, CD21, PD-1, CD68, and DAPI). Subsequently, sections underwent automated cell scoring and analysis of spatial interactions, defined as cells co-occurring within 30 μm. Shannon’s entropy, a metric describing species biodiversity in ecological habitats, was applied to quantify immune infiltrate diversity of cell types and spatial interactions. Immune infiltrate diversity indices were tested in multivariable Cox regression and Kaplan–Meier analysis for overall (OS) and progression-free survival (PFS). Results: Increased diversity of cell types (HR = 0.19 95% CI 0.06–0.65, p = 0.008) and cell spatial interactions (HR = 0.39, 95% CI 0.20–0.75, p = 0.005) was associated with favourable OS, independent of the Follicular Lymphoma International Prognostic Index. In the rituximab-treated subset, the favourable trend between diversity and PFS did not reach statistical significance. Conclusion: Multiplex immunofluorescence and Shannon’s entropy can objectively quantify immune infiltrate diversity and generate prognostic information in FL. This automated approach warrants validation in additional FL cohorts, and its applicability as a pre-treatment biomarker to identify high-risk patients should be further explored. The multiplex image dataset generated by this study is shared publicly to encourage further research on the FL microenvironment

Tumour Infiltrating Lymphocytes in Follicular Lymphoma - additional data

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