PMC5944263_01
Patient #006 timeline and immune infiltrate analysis in post-vaccination subcutaneous metastasis (SC mts). (A) The patient was randomized to the CSF-470 vaccine arm of CASVAC-0401 study. Tumor resections of primary tumor, sentinel node, and SC mts are indicated by triangles. Peripheral blood mononuclear cell (PBMC) samples were obtained at 0 (PBMC-PRE), 6 (PBMC-POST-1), 12 (PBMC-POST-2), 24 (PBMC-POST-3), and 48 months (PBMC-POST-4) from vaccine protocol. *Lung and SC mts were detected at the same time; tumor-infiltrating lymphocytes (TIL) were obtained from the SC mts. Samples selected for T-cell receptor beta sequencing are indicated with red triangles and in italics. (B) Representative pictures of the same region in the SC mts after IHC staining for CD8, Granzyme-B (GZMB), in situ nick translation (ISNT), CD4, PD-1, and PD-L1 are shown (DAB, brown). Insets show positive staining under high magnification (1,000x). Scale bars = 100 microm.
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Three months later, an axillary sentinel lymph node with micrometastasis was excised (January 2011) (Figure 1A). The primary tumor in the back was a nodular, epithelioid CM with 3.0 mm Breslow thickness, micro-ulceration, vessel infiltration by tumor cells, and 15% Ki-67+ tumor proliferating cells (Figure S1I in Supplementary Material). The SC mts that developed in the patient's back, in the vicinity of the previous scar, had defined expansive edges, with areas of epithelioid tumor cells adjoined by zones of dense fibrosis and zones fulfilled with brisk lymphocyte infiltration (Figure S1II in Supplementary Material). In the areas of brisk lymphocyte infiltration, necrotic/apoptotic tumor cells, distinguished by cell shrinkage with nuclear disruption, were observed surrounded by immune cells, mainly CD8+ and CD4+, some of them firmly attached (Figure 1B); Granzyme-B staining revealed positive cytosolic granules in tumor cells. In situ nick translation (ISNT) detected positive nuclei and vesicles within dying-tumor cells, confirming the presence of apoptotic tumor cells in close contact with lymphocytes (Figure 1B). Few PD-1+ lymphocytes were observed attached to tumor cells, PD-L1 expression was negative (Figure 1B). TCRbeta repertoire genes, since within the TCR, this region contains the highest variability, making it a suitable molecular identifier for tracking individual T-cell clones. TCRbeta sequence analyses were performed in the SC mts TIL and in PBMC samples obtained before (PBMC-PRE), during CSF-470 immunization (PBMC-POST-1 and PBMC-POST-3), and 2 years after completing the vaccine protocol (PBMC-POST-4) (Figure 1A; Tables S2-S6 in Supplementary Material).
Immune profiling of patient #006. (A) Representative pictures of the primary tumor and subcutaneous metastasis biopsies infiltrated by CD8+, CD4+, CD20+, and Foxp3 lymphocytes determined by IHC (DAB, brown). (B) After counting total biopsy area, immune-to-tumor cell ratios were determined for each tumor biopsy as well as the relative ratio between primary tumor and SC metastasis (1). (C) Absolute counts of total lymphocytes, T CD4+, T CD8+, Treg, and NK cells from PBMC were determined throughout CASVAC-0401 treatment and follow-up by flow cytometry; also, absolute counts of lymphocytes (Ly) and neutrophils (Neu) are shown (D). Scale bars = 50 microm.
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Comparative analysis of the immune cells infiltrating the primary tumor and the SC mts revealed an increase mainly of CD8+, CD4+, and CD20+ lymphocytes (Figure 2A). CD8+, CD4+, and CD20+ lymphocytes increased 10-fold, 3-fold, and 2-fold, respectively, in the SC mts with respect to the primary tumor, while Foxp3+ cells were scarce (Figure 2B). CD11c+ and CD68+ antigen (Ag)-presenting cells were also frequently detected; CD45Ro+ memory cells decreased at the SC mts (Figure 2B). Peripheral blood lymphocyte analyses revealed a sustained increase in total lymphocytes, and of T CD4+, T CD8+, and NK cells during CSF-470 treatment (Figure 2C). T cells increased by the end of the protocol (24 months); low and stable levels of Tregs were observed throughout treatment (Figure 2C). Two years after completing the immunization protocol, CD4+ increased while CD8+ levels decreased, although remaining at higher levels than at baseline; NK and Tregs stayed still (Figure 2C). Neutrophils/lymphocytes ratio sharply diminished during CSF-470 immunization (Figure 2D). Analysis of mutational signature revealed fingerprints related to failure of DNA double-strand break-repair by homologous recombination; large numbers of CC>TT dinucleotide mutations at dipyrimidines due to ultraviolet light exposure, typical of melanoma, were also found (Figure S2 in Supplementary Material). An increase in CD4+ and CD8+ lymphocytes in the peripheral compartment after immunization with the CSF-470 vaccine, as well as a brisk infiltration of CD4+ and CD8+ lymphocytes in the SC mts, was observed (Figure 2A).
Distribution and tracking of the T-cell receptor beta (TCRbeta) immune repertoire in peripheral blood mononuclear cells (PBMC) and subcutaneous metastasis from patient #006. (A) Percentage of total TCRbeta clones covered by the TOP100 clones, the 100 most frequent ones (nucleotide sequences). (B) Total TCRbeta clones (%), from highest to lowest, in function of their cumulative frequency (nucleotide sequences). (C) The proportion of TCRbeta clones (%) that constitute the TOP-25 or TQ clones, and the TOP-50 clones are shown (TOP-25/50, the proportion of clones that make up the top cumulative frequency of 25/50). (D) Pattern distribution of TCRbeta clones in total PBMC, total tumor-infiltrating lymphocytes (TIL), and Top-Quartile TIL (TQ-TIL); the number of clones for each pattern and total clones analyzed in each case is indicated. Patterns: 1, present at baseline, increases in time; 2, absent at baseline, increases in time; 3, absent at baseline, detected at POST-3 sample; 4, present at baseline, decreases in time; 5, present at baseline, absent in time; and 6, absent at blood, present in tumor. (E) Common sequences among PBMC-PRE, PBMC-P1, PBMC-P3, and TIL samples. (F) The proportion of TIL clones present in blood at different times. (G) Venn diagram illustrating common clones among TIL and POST-4 samples regarding amino acid sequences. (H) Distribution in tracking patterns of total and TQ-TIL clones shared with PBMC POST-4. Pattern 3: absent at baseline, detected at POST-4 sample.
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TCRbeta clones (TOP100) indicated an increment in their cumulative frequency throughout immunization, supporting preferential expansion of a subpopulation of T-cell clones (Figure 3A). TOP100 TIL clones from the SC mts showed an oligoclonal TCRbeta repertoire almost attaining the top-quartile most frequent clones (TQ) (Figure 3A). Also, the frequency of each TCRbeta clone in the immune repertoire (%), ordered from highest to lowest, was plotted in function of its cumulative frequency (%) to compare blood samples (Figure 3B). A similar pattern was observed for PBMC-POST-4 (Figure 3B). When analyzing the proportion of clones that make up the top cumulative frequency of 25 [TOP-25 or Top-Quartile (TQ)] or 50 (TOP-50), it decreases over time; further supporting the expansion of a subpopulation of T-cell clones (Figure 3C). To further analyze the changes induced in the TCRbeta immune repertoire of patient #006 throughout immunization with the CSF-470 vaccine, six different patterns for tracking T-cell clones in blood were defined (Figure 3D; Figure S3A in Supplementary Material). Analysis of shared amino acid sequences among TIL and blood samples revealed that all samples shared 10% of the clones, and 16% were shared by TIL and PBMC-POST3 (Figure 3E). When total TIL clones were analyzed considering the tracking patterns for peripheral clones, 53% of TIL clones were only detected at the tumor microenvironment (pattern 6); 13.6% corresponded to preexisting clones that expanded during immunization (pattern 1), clones emerging during immunization and detected at 12 months (2.2%, pattern 2) and 24 months by the end of immunization (15.9%, pattern 3) (Figure 3D). 9.3 and 6.0% of TIL clones diminished their frequency in blood or were no longer detected. TIL clones (TQ-TIL), preexisting clones (pre-vaccination) were enriched (pattern 1, 31.6%), prevailing along with late-emerging clones (pattern 3, 19.8%) (Figure 3D). 58% of TQ-TIL clones corresponded to pattern 1 clones that have expanded in blood at least three times; and 45% of TQ-TIL clones were in the 2.4% most abundant clones in PBMC-POST-3 sample (pattern 3) (data not shown). This supports the idea that a considerable proportion of abundant TIL clones (TQ-TIL) are among those that were expanded in blood, either preexistent or emerging post-vaccination. TQ-TIL clones detected only at the tumor microenvironment accounted for 31.6% (pattern 6) (Figure 3D). TIL clones detected in blood doubled from 5% (PBMC-PRE) to 10.7% total frequency (PBMC-POST-3) (Figure 3F). Two years later (PBMC-POST-4 sample), TIL clones were still detected in blood with a cumulative frequency of 8.9% (Figure 3F). TIL clones were shared with the POST-4 sample (Figure 3G). These 706 common clones were distributed mainly in four patterns, 56% of them corresponded to patterns 1 and 2; while 21.6% were detected only at POST-4 blood sample (pattern 3) (Figure 3H; Figure S3 in Supplementary Material). TQ-TIL clones shared with PBMC-POST-4, expanded preexisting and clones arising during immunization accounted for 70.8% (patterns 1 and 2) (Figure 3H). These results suggest that CSF-470 vaccination in patient #006 modulated the repertoire of T-cell clones and their frequency, and that particularly those clones expanded during immunization persisted at least 24 months after the end of the vaccination protocol.