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Inoculation site from a cutaneous melanoma patient treated with an allogeneic therapeutic vaccine: a case report.

ABSTRACT: We have developed a therapeutic vaccine consisting of a mixture of lethally-irradiated allogeneic cutaneous melanoma cell lines with BCG and GM-CSF as adjuvants. The CSF-470 vaccine is currently being assayed in a Phase II-III trial against medium-dose IFN-?2b. All vaccinated patients immunized intradermally developed large edematous erythema reactions, which then transformed into subcutaneous nodules active for several months. However, vaccine injection sites were not routinely biopsied. We describe the case of a female patient, previously classified as stage III, but who, due to the simultaneous discovery of bone metastases only received one vaccination was withdrawn from the study, and continued her treatment elsewhere. This patient developed a post-vaccination nodule which was surgically removed 7?weeks later, and allowed to analyze the reactivity and immune profiling of the inoculation site. An inflammatory reaction with zones of fibrosis, high irrigation, and brisk lymphoid infiltration, primarily composed of CD8(+) and CD20(+) lymphocytes, was observed. There were no remaining BCG bacilli, and scarce CD4(+) and Foxp3(+) T cells were determined. MART-1 Ag was found throughout the vaccination site. CD11c(+) Ag presenting cells were either dispersed or forming dense nests. Some CD11c(+) cells proliferated; most of them contained intracellular MART-1 Ag, and some interacted with CD8(+) lymphocytes. These observations suggest a potent, long-lasting local inflammatory response with recruitment of Ag-presenting cells that incorporate melanoma Ags, probably leading to Ag presentation to naïve T cells.

SUBMITTER: Aris M

PROVIDER: S-EPMC4378302 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Inoculation site from a cutaneous melanoma patient treated with an allogeneic therapeutic vaccine: a case report.

Aris Mariana M Bravo Alicia Inés AI Barrio María Marcela MM Mordoh José J

Frontiers in immunology 20150330

We have developed a therapeutic vaccine consisting of a mixture of lethally-irradiated allogeneic cutaneous melanoma cell lines with BCG and GM-CSF as adjuvants. The CSF-470 vaccine is currently being assayed in a Phase II-III trial against medium-dose IFN-α2b. All vaccinated patients immunized intradermally developed large edematous erythema reactions, which then transformed into subcutaneous nodules active for several months. However, vaccine injection sites were not routinely biopsied. We des ...[more]

PMID: 25870600

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Project description:The CSF-470 vaccine consists of lethally-irradiated allogeneic cells derived from four cutaneous melanoma cell lines administered plus BCG and GM-CSF as adjuvants. In an adjuvant phase II study vs. IFN-α2b, the vaccine significantly prolonged the distant metastasis-free survival (DMFS) of stages IIB-IIC-III melanoma patients with evidence of the induction of immune responses against vaccine cells. Purpose: The aim of this study was to analyze the antigens against which the immune response was induced, as well as the T-helper profile and lytic ability of immune cells after CSF-470 treatment. Methods: HLA-restricted peptides from tumor-associated antigens (TAAs) were selected from TANTIGEN database for 13 evaluable vaccinated patients. In addition, for patient #006 (pt#006), tumor somatic variants were identified by NGS and candidate neoAgs were selected by predicted HLA binding affinity and similarity between wild type (wt) and mutant peptides. The patient's PBMC reactivity against selected peptides was detected by IFNγ-ELISPOT. T-helper transcriptional profile was determined by quantifying GATA-3, T-bet, and FOXP3 mRNA by RT-PCR, and intracellular cytokines were analyzed by flow cytometry. Autologous tumor cell lysis by PBMC was assessed in an in vitro calcein release assay. Results: Vaccinated patient's PBMC reactivity against selected TAAs derived peptides showed a progressive increase in the number of IFNγ-producing cells throughout the 2-yr vaccination protocol. ELISPOT response correlated with delayed type hypersensitivity (DTH) reaction to CSF-470 vaccine cells. Early upregulation of GATA-3 and Foxp3 mRNA, as well as an increase in CD4+IL4+cells, was associated with a low DMFS. Also, IFNγ response against 9/73 predicted neoAgs was evidenced in the case of pt#006; 7/9 emerged after vaccination. We verified in pt# 006 that post-vaccination PBMC boosted in vitro with the vaccine lysate were able to lyse autologous tumor cells. Conclusions: A progressive increase in the immune response against TAAs expressed in the vaccine and in the patient's tumor was induced by CSF-470 vaccination. In pt#006, we demonstrated immune recognition of patient's specific neoAgs, which emerged after vaccination. These results suggest that an initial response against shared TAAs could further stimulate an immune response against autologous tumor neoAgs.

Project description:The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFN?2b 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 ? (TCR?) 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 TCR? 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.

Project description:BackgroundCutaneous melanoma is the skin cancer with the highest mutational burden and metastatic rate. Early genetic alterations and biomarkers of distant progression are a point of interest. In addition to germline-susceptibility loci, almost 30% of melanomas arise from precursor benign nevi lesions, providing a source for malignant transformation.Case presentationPatient#009 developed a cutaneous melanoma over a nevus, followed by progression to regional and distant metastases in months, unresponsive to targeted therapy. To search for the genetic contribution to this rapid progression, a longitudinal analysis was performed through WES of germline, nevi, primary tumor, and a metastatic lymph node. Differential SNP/INDEL and CNV gene alterations, with functional impact on key pathways and cancer hallmarks in each step of evolution, were discerned. Tumor-associated nevus was, for the first time, split into two sections, distant and adjacent to the primary tumor, to study its heterogeneity. Shared SNP alterations, with stable allele fraction from germline to metastasis were detected, mainly affecting DNA repair genes and promoting genome instability. Early somatic alterations, shared by nevi and primary and metastatic tumors, included BRAFV600E and focal copy-loss of several genes, acquiring additional cancer hallmarks. Phylogenetic analyses revealed that these common somatic alterations would provide a "bridge", allowing progression from a benign to a malignant state. Distant and adjacent nevi were rich in alterations, presenting differential SNP and CNV alterations. Upon tumor transformation, a marked increase in CNV over SNP alterations was determined. Both the number of SNP and CNV-affected genes, including known driver genes, increased throughout progression, although TMB levels remained lower than expected for melanoma. Typical alterations in BRAFV600E tumors related to intrinsic resistance to targeted therapy were found, including BRAF amplification and loss of PTEN, CDKN2A/B, and TP53 surveillance genes. Finally, numerous metastatic alterations were detected, further promoting tumor progression.ConclusionsIn this patient, longitudinal WES analysis revealed a sequential and cumulative pattern of genetic alterations, where germline and nevi somatic events contributed early to its rapid clinical progression. In this case report, we found tumor-associated nevi as genetically heterogeneous precursor entities, in which potential prognostic biomarkers should be studied prospectively.

Dataset Information

Inoculation site from a cutaneous melanoma patient treated with an allogeneic therapeutic vaccine: a case report.

Publications

Inoculation site from a cutaneous melanoma patient treated with an allogeneic therapeutic vaccine: a case report.

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