At advanced stages, many types of cancer can infiltrate the pleural cavity, resulting in a collection of cancer-containing fluid termed malignant pleural effusion (MPE). MPEs are marked by persistent inflammation that negatively affects the quality of life for the patient and are often the herald of the terminal stages of cancer. MPEs possess a large collection of immune cells that may contain dormant effector cells, which could be expanded on for therapy. However, knowledge of the phenotypic composition of these collections and how they interact is limited.
In a recently published paper, researchers at the Pittsburgh School of Medicine used a combination of RNA-seq and targeted immune sequencing to link a protein overexpressed within MPEs to reduced levels of T cell receptor (TCR) diversity1.
The role of the immune repertoire in cancer progression
The diversity of T cell and B cell receptors, collectively referred to as the adaptome, is a hallmark feature of adaptive cellular immunity and is what allows it to respond to a myriad of pathogenic and endogenous threats. Decreased T cell diversity is a common setting of cancer and may lead to reduced protection from tumor cells and other threats expressing neoantigens. Furthermore, stronger T cell diversity is associated with better patient response to immunotherapy.
In order to understand how the immune adaptome is affected in MPEs, Soloff et al utilized high-throughput immune repertoire sequencing using iRepertoire’s dimer avoided Multiplex PCR (dam-PCR) for simultaneous amplification of all seven B cell and T cell receptor chains in one reaction. In addition to amplifying all seven chains, dam-PCR incorporates unique molecular identifiers, enabling an extremely quantitative analysis of the entire B and T cell repertoire.
Simultaneous sequencing of the entire immune repertoire
Soloff et al extracted RNA from MPEs in 14 patients and amplified them into a library for Next-Generation Sequencing (NGS). The whole library preparation process was fully automated in the iR-Processor and iR-Cassette. Using dam-PCR, paired-end sequencing was performed for an average read depth of 30,000 reads per sample before readouts were analyzed in iR-map and visualized in iRweb.
With the quantitative immune sequencing power of dam-PCR, Soloff et al were able to calculate the diversity of the TCR repertoire for each patient sample. They found that patients with high levels of HMGB1, a multifaceted nuclear protein overexpressed in lung cancer MPEs, had significantly diminished diversity indices for TCR diversity. The role of HMGB1 in influencing immune cell phenotypes implicates its role in tumorigenesis within MPEs and suggests that HMGB1 may serve as a viable therapeutic target.
MPEs typically only appear during the late stages of advanced cancer and are typically characterized by an abundance of immune cells. This condition results in a persistent inflammatory effect, which is believed to play a part in immune evasion and immune dysfunction. However, our understanding of the phenotypes of these immune cells, their tumor specificity, and how they are impacted by the MPE is not well known.
Adding immune repertoire sequencing via dam-PCR provides the ability to study the unique responses of a patient’s immune system in the context of late-stage cancers where constant inflammatory responses are a major feature. Understanding the composition of these immune cells and how they interact with other proteins could open new therapeutic approaches in treating late-stage cancers.
- Soloff, A.C. et al. HMGB1 Promotes Myeloid Egress and Limits Lymphatic Clearance of Malignant Pleural Effusions. Frontiers in Immunology 11, (2020). https://doi.org/10.3389/fimmu.2020.02027