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. 2013 Dec;231(4):433-440.
doi: 10.1002/path.4260.

High-throughput Sequencing of T-cell Receptors Reveals a Homogeneous Repertoire of Tumour-Infiltrating Lymphocytes in Ovarian Cancer

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Free PMC article

High-throughput Sequencing of T-cell Receptors Reveals a Homogeneous Repertoire of Tumour-Infiltrating Lymphocytes in Ovarian Cancer

Ryan O Emerson et al. J Pathol. .
Free PMC article

Abstract

The cellular adaptive immune system mounts a response to many solid tumours mediated by tumour-infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma, with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood, including specificity, clonality, and spatial heterogeneity of the T-cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T-cell development, the TCR beta chain sequence serves as a molecular tag for each T-cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumours and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumour and are distinct from the circulating T-cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T-cell compartment of peripheral blood.

Keywords: T cells; high-throughput sequencing; ovarian carcinoma; tumour heterogeneity; tumour-infiltrating lymphocytes (TILs).

Figures

Figure 1
Figure 1. Schematic of sample collection
(A) After debulking, a one centimeter slice was taken from the center of the primary and metastatic tumor along the longest diameter. These middle slices were designated Slice A from the metastasis and Slice C from the primary tumor. Additional 1-cm slices from either side of the middle slice were taken when possible from the primary tumor (slices B and D). (B) These slices were aligned with a grid allowing for up to 25 biopsy punches to be taken from each slice with a 2-cm spacing from the center of one punch to the center of the adjacent punch. Additionally, four punches were taken around the periphery of the tumor slice at 12, 3, 6 and 9 o'clock (punches 26, 27, 28 and 29). A peripheral blood sample was also collected from which mononuclear cells were isolated.
Figure 2
Figure 2. T cell infiltration in five ovarian carcinomas
We used a droplet digital PCR assay to determine the level of T cell infiltration in patient samples. Among tumor tissue samples from the major site of each patient's malignancy (primary tumor in patients 2 and 5; metastatic tumor in patients 1, 3 and 4), we calculated the proportion of T cells for each patient among nucleated cells (mean ± SEM; N= the number of different tissue cores examined for each patient). With the exception of patients 1 and 2, whose tumors have very similar T cell numbers, each pair of tumors has significantly different level of T cell infiltration (n = 9 pairwise comparisons, p < 0.01 by two-tailed T test in each case).
Figure 3
Figure 3. TCRB repertoire overlap across a large metastatic tumor slice
We calculated TCRB repertoire overlap between the central punch (13) of a large tumor slice from patient 3 and all other samples from the same patient, including one peripheral blood sample and one primary tumor sample. The spatial organization of the metastatic tumor punches is shown and each sample is colored according to its degree of TCRB repertoire overlap with the central punch. Other punches from the metastatic tumor in this patient share high TCRB repertoire overlap (0.75), with no obvious relationship between TCRB repertoire overlap and distance from the center punch (R2 = 0.014). TCRB repertoire overlap is somewhat lower in the primary tumor punch (0.57), and considerably lower in peripheral blood (0.19). All comparisons are marked on the color scale at bottom. Quartile ranges are shown for the metastatic punches.
Figure 4
Figure 4. Within and between-sample TCRB repertoire overlap and variability
A) Genomic DNA from four tissue punches from a metastatic tumor (patient 4) was extracted and PCR and sequencing reactions were performed in duplicate for each sample. Measurements were made to calculate the within-tissue sample comparison (red lines, n = 4; which should be influenced only by technical error and TCRB repertoire sampling error) and between-tissue sample comparisons (blue lines, n = 24; which should be affected by technical error, sampling error and spatial heterogeneity). B) TCRB repertoire overlap between each pair of samples was calculated, and we compared the mean TCRB repertoire overlap (+/− Standard Error of the Mean) of within-sample and between-sample comparisons. A p-value was calculated using a two-tailed unpaired t-test. Duplicate samples do not have systematically higher TCRB repertoire overlap, indicating that TCRB repertoire overlap values are primarily influenced by technical and sampling error rather than spatial location within the tumor.
Figure 5
Figure 5. Clustering analysis of TCRB repertoire overlaps among patients
For each of four patients we calculated pairwise distances among all tissue samples using TCRB repertoire overlap to create a distance metric (see Methods). These distances were used to construct a neighbor-joining tree for each patient. Trees are color-coded by tissue source: primary tumor (green); metastatic tumor (blue); peripheral blood (red). The lack of substantial branching structure (i.e., star phylogeny) observed among tumor samples from all patients indicates spatial homogeneity among tumor samples, while the larger distance between tumor and blood samples indicates that tumor samples share considerably more TCRB sequences with each other than with peripheral blood.

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