Abstract
The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCRβ repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c+ nested-clusters, brisk CD8+ and scarce FOXP3+, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages. A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8+PD1+ T-cells, CD20+ B-cells, and scarce FOXP3+ cells. Increasing DTH score and IFN-γ ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization. TCRβ repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well. We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.
Highlights
Treatment of cutaneous melanoma (CM) has greatly improved in recent decades due to two main discoveries
One of the unanswered questions about repeated vaccinations with CSF-470 plus Bacillus Calmette Guerin (BCG) and rhGM-CSF is the cellular composition of the VAC-SITE, since their systematic analysis was not contemplated per protocol
Large numbers of Langhans multinucleated giant cells (LMGC) were observed, some of them with more than 10 nuclei, which showed a typical circular peripheral arrangement (Figures 1H,I); no BCG bacilli were observed within these cells or elsewhere
Summary
Treatment of cutaneous melanoma (CM) has greatly improved in recent decades due to two main discoveries. In a previous analysis of vaccinated patient-006 from the CASVAC-0401 trial, an increase in tumor infiltrating lymphocytes (TIL) in a cutaneous metastasis (CMTS) developing at the end of the immunization protocol was observed. This lesion was infiltrated with a T-cell repertoire that expanded in blood over the 2-yr treatment, persisting 2yr after completing the protocol; along with lymphocytes only detected at the tumor site [16]. As well as the surgical events and blood extractions, are indicated (Supplementary Figure 2)
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