Changes in the TCRβ Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report

Abstract

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor β (TCRβ) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRβ repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

Keywords: CSF-470 vaccine; T-cell receptor β immune repertoire; cancer immunogram; cutaneous melanoma; tumor immune infiltration.

Figures

Figure 1

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 β 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 (1,000×). Scale bars = 100 µm.

Figure 2

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 µm.

Figure 3

Distribution and tracking of the T-cell receptor β (TCRβ) immune repertoire in peripheral blood mononuclear cells (PBMC) and subcutaneous metastasis from patient #006. (A) Percentage of total TCRβ clones covered by the TOP100 clones, the 100 most frequent ones (nucleotide sequences). (B) Total TCRβ clones (%), from highest to lowest, in function of their cumulative frequency (nucleotide sequences). (C) The proportion of TCRβ 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 TCRβ 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.