Serial FNA allows direct sampling of malignant and infiltrating immune cells in patients with B-cell lymphoma receiving immunotherapy

Abstract

Background: Fine-needle aspiration (FNA) is used to diagnose malignancies, recurrences, and metastases. The procedure is quick and well tolerated and can be facilitated by ultrasound guidance.

Methods: This article describes the authors' experience in using serial FNA to harvest cellular material during 4 clinical trials of immunotherapy by in situ vaccination in patients with low-grade lymphoma.

Results: Two hundred ninety-six FNA samples were collected from 44 patients over a span of approximately 6 weeks for each patient. Samples were sufficient in quantity and quality to be analyzed by flow cytometry and/or single-cell messenger RNA sequencing. FNA samples yielded an average of 12 × 106 cells with a mean cellular viability of 86%. Material collected from the tumor lymph nodes differed significantly in the proportions and phenotypes of cellular populations in comparison with matched peripheral blood samples. A comparison of flow cytometry results obtained by FNA directly from the patient and by FNA performed ex vivo and a dissociation of the same lymph node after surgical excision confirmed that FNA sampling of the patient accurately represented the tumor and the microenvironment. An analysis of the FNA samples from immunotherapy-treated target lymph nodes versus nodes from nontreated tumor sites provided insight into the impact of specific immunotherapy regimens.

Conclusions: This is the largest study describing the use of serial FNA sampling to harvest cellular material during immunotherapy clinical trials. The success of this technique opens the door for FNA sampling to expand significantly future investigations of the dynamic effects of investigational agents, be they immunotherapies or targeted therapies.

Trial registration: ClinicalTrials.gov NCT02927964 NCT03410901 NCT02266147 NCT02254772.

Keywords: clinical trial; fine-needle aspiration (FNA); immunotherapy; lymphoma.

Conflict of interest statement

Conflict of Interest Disclosures: The authors made no disclosures.

© 2021 American Cancer Society.

Figures

Figure 1.

Overview of FNA methodology in clinical trials. Two FNA sites were identified in each patient including “Site A”, the treated site, and “Site B”, the non-treated site. Following pre-treatment CT scans, an initial pre-treatment FNA was performed at sites A and B. Low-dose radiation therapy followed by the first immunotherapy dose was then administered to Site A only. Eight days later, a second FNA was performed at sites A and B (Week 2). A third FNA was performed at sites A and B at week 6, subsequent to the final immunotherapy dose. CT scans were performed at intervals defined by each trial protocol. Each trial utilized different immune agonists or combinations of immune agonists.

Figure 2.

Visualization of high-dimensional flow cytometry data analyzed by the dimensional-reduction algorithm, viSNE. Cells are clustered by surface phenotypes: CD3+ T cells (blue), CD56+ NK cells (orange), CD14+ macrophages or monocytes (purple) and CD33+ granulocytes (brown). CD19+ B cells are split between Lambda- tumor cells (green) and Lambda+ normal B cells (red). (A) Example of 2 separate FNA from 2 different sites of a follicular lymphoma patient. (B) Single-cell suspension of a surgical biopsy from the same patient. (C) FNA performed on the biopsy ex vivo and prior to dissociation. (D) Example of peripheral blood drawn at the same time as the FNA.

Figure 3.

Correlation of 3 major cell populations found in the excisional biopsies and FNA performed on 4 different patients. All biopsies and corresponding FNA were performed prior to treatment and within 8 days of each other. Data is expressed as percent of all live cells. Each color represents a different patient and each symbol represents a different cell population as noted in the legend.

Figure 4.

Fine needle aspirates yield high quality single cell sequencing data. (A) Dimensionality reduction plot showing cells clustered by single-cell gene expression profiles. Cells from 3 fine needle aspirates from the same tumor from 3 different timepoints from one patient are combined and clustered as one dataset. (B) Frequencies of tumor T cell populations before and after treatment. PRE, pre-treatment. WK2, week 2 post-treatment. WK6, week 6 post-treatment. Tfh cells, T follicular helper. Treg, Regulatory T cells. NK/NKT cells, natural killer/natural killer T cells.