T-cell receptor profiling in cancer

Abstract

Immunosequencing is a platform technology that allows the enumeration, specification and quantification of each and every B- and/or T-cell in any biologic sample of interest. Thus, it provides an assessment of the level and distribution of all the clonal lymphocytes in any sample, and allows "tracking" of a single clone or multiple clones of interest over time or from tissue to tissue within a given patient. It is based on bias-controlled multiplex PCR and high-throughput sequencing, and it is highly accurate, standardized, and sensitive. In this review, we provide evidence that immunosequencing is becoming an important analytic tool for the emerging field of immune-oncology, and describe several applications of this approach, including the assessment of residual disease post therapy in lymphoid malignancies, the prediction of response to immunotherapeutics of solid tumors containing tumor infiltrating lymphocytes, the identification of clonal responses in vaccination, infectious disease, bone marrow reconstitution, and autoimmunity, and the exploration of whether there are population-based stereotyped responses to certain exposures or interventions.

Keywords: Ig/TCR sequence biomarkers; Immunosequencing; Lymphoid clonality assessment/tracking.

Figure 1

The basis of the immunosequencing assay. Multiple V, D and J gene segments exist in the germline genome. Initial receptor diversity is generated by recombination of V, D and J segments; and additional non‐templated diversity is introduced at the junctions by insertion of random nucleotides (N). The immunosequencing assay uses a multiplex PCR with forward primers for each V segment and reverse primers for each J segment. A simplified TCRB locus is shown as an example.

Figure 2

The use of immunosequencing allows the tracking of index sequences as well as murine‐derived CART‐19 vector sequences at different timepoints. Genomic DNA extracted from a bone marrow sample at the time of diagnosis was provided by the ordering physician. Sample chain of custody and method of DNA extraction is on file. Two index sequences were identified from this patient at the diagnostic time point (green and blue circles). Both index sequences decreased in frequency more than 100X in the next follow‐up sample (post‐infusion time point #1), which was quite B‐cell depleted. In comparison, both index sequences showed an approximately 10X increase in frequency in the later sample (post‐infusion time point #2). The dominant sequence that emerged in post‐infusion time point #1 (yellow square), which was not present in the diagnostic sample, shows a marked decrease in post‐infusion time point #2. This clone was recognized as a murine sequence associated with the CART‐19 vector, and was identified because of fortuitous cross‐reactivity with human IgH primers. Thus, its reported sequence does not constitute a rigorously quantitative assessment of its presence. The navy and the orange lines indicate the stated limit of detection of malignant cells by flow cytometry and light microcopy, respectively.

Figure 3

Effects of CTLA‐4 blockade on the T‐cell repertoire analyzed by immunosequencing. Tremelimumab treatment does not significantly change the clonality (A) or the diversity (B) of the circulating T‐cell repertoire as a whole (green bars). However, when patients are classified by the absence (blue) or presence (orange) of toxicity events, there is a clear trend for decreased clonality and increased diversity that associates with toxicity. The absolute number of unique sequences also showed a positive correlation with toxicity (C). All error bars indicate the standard error of the mean. Adapted from Robert et al. (2014) Clin. Cancer Res. 20:2424.

Figure 4

Immunosequencing reveals a homogeneous distribution of TILs. The TCRB repertoire is highly homogeneous over the mass of the metastatic tumor site (orange). Also, the overlap between the metastatic site and the primary tumor (green) and is much higher than that between the tumor and a paired blood sample (blue). Adapted from Emerson et al. (2013b) J. of Pathology 231:433–440.

Figure 5

Quantitative sequencing of TCRB in patients with melanoma. Progressors (blue) were associated with both a lower number of TILs (shown as percentage TIL infiltrate) and a lower TIL clonality than responders (orange). Adapted from Tumeh et al. (2014) Nature 515(7528):568–571.