Cryoablation and post-progression immune checkpoint inhibition in metastatic melanoma: a phase II trial

Abstract

Image-guided percutaneous cryoablation is an established minimally invasive oncologic treatment. We hypothesized that cryoablation may modify the immune microenvironment through direct modulation of the tumor, thereby generating an anti-tumor response in tumors refractory to immune checkpoint inhibition (ICI). In this non-randomized phase II single-center study (NCT03290677), subjects with unresectable melanoma progressing on ICI underwent cryoablation of an enlarging metastasis, and ICI was continued for a minimum of two additional cycles. The primary endpoints were safety, feasibility and tumor response in non-ablated lesions. From May 2018 through July 2020, 17 patients were treated on study. The study met its primary endpoints with the combination strategy found to be safe and feasible with an objective response rate of 23.5% and disease control rate of 41% (4 partial response, 3 stable disease). Our data support further study of this synergistic therapeutic approach.

Fig. 1. Melanoma study design and clinical response to combination cryoablation and ICI.

A Study schema. Visual production credits “iStock.com/DennyFachrulRozzy” and iStock.com/MaskaRad”. B Waterfall plot of best response/change in tumor burden from baseline post-cryoablation (n = 17). Source data are provided as a Source Data file. C Computed tomography of one patient with a durable response on trial (n = 1). Axial maximum intensity projection chest computed tomography (CT) of a 64-year-old male with progression of metastatic melanoma on pembrolizumab monotherapy. Baseline CT performed three months prior to cryoablation shows a 28 mm metastasis in the right lower lobe (RLL, panel 1, arrow), an 11 mm metastasis in the left upper lobe (LUL, panel 2, arrow), and right hilar lymphadenopathy (panel 3, arrow). CT images obtained to guide cryoablation of the dominant RLL metastasis demonstrate interval enlargement of the target (40 mm, panel 4, arrow), LUL metastasis (13 mm, panel 5, arrow), and right hilar lymphadenopathy (panel 6, arrow), and a new subpleural 8 mm RLL metastasis (panel 5, circle). Three-, six-, and nine-month follow-up CT shows resolution of the subpleural RLL non-ablated metastasis (panels 8, 11, 14), shrinkage of the LUL non-ablated metastasis to 7 mm, 4 mm, and 3 mm (panels 8, 11, 14, arrow), and resolution of right hilar lymphadenopathy (panels 9, 12, 15, arrow). The ablated RLL metastasis also decreased in size (panels 7, 10, 13, arrow), respectively. CT, computed tomography; LUL, left upper lobe; RLL, right lower lobe. D Duration of ICI pre- and post-cryoablation (n = 17). Cryoablation occurred on day 0. Source data are provided as a Source Data file.

Fig. 2. Single-cell RNA-sequencing overview and cell composition analysis of ICI-resistant melanoma tumors treated with cryoablation.

A Overview of the scRNA-seq cohort. Visual production credits “iStock.com/MaskaRad”. B Summary of available samples by patient and response status, organized by anatomical site and labeled by timing obtained (i.e pre-tx: pre-ablation, pre+post tx: pre-ablation and post-ablation). C Uniform manifold approximation and projection (UMAP) plot of all single cells passing quality control filtration, colored by cell lineage. D UMAP expression density plots of the following lineage-defining marker genes (left to right, top to bottom): PMEL, VWF, CD3E, LYZ, CD79A, COL1A1. E Box plots displaying (top) the percentage of T, B/plasma, and myeloid cells out of all CD45+ cells, and (bottom) the percentage of melanocyte, endothelial, and stromal cells out of CD45- cells for pre-treatment samples only. Box plots are grouped by ultimate response to cryoablation, comparing patients with progressive disease (n = 10) to those without (n = 3). Box plots indicate interquartile range, with horizontal line indicating median, and whiskers extending to maxima and minima excluding outliers (>1.5 * IQR). Indicated p values are for the Kruskal–Wallis test, uncorrected. PD progressive disease, SD stable disease, PR partial response. Source data for Fig. 2 are provided as a Source Data file.

Fig. 3. Immune cell type-specific gene transcription associated with response to cryoablation therapy.

A UMAP of T cell subclustering with cluster annotations. B Box plots displaying the frequency of T cells belonging to a specific subcluster per patient comparing PD (n = 9), SD (n = 2), and PR (n = 2). Horizontal line indicates median. C Plots of DESeq2-normalized counts for highlighted genes differentially expressed by T cells between categories of best response post-cryoablation for pre-treatment samples (n = 13). FDR-adjusted p-values are for two-sided DESeq2 Wald test differential expression results; p_benefit denotes grouping SD + PR. D Gene set enrichment analysis (GSEA) for the differentially expressed T cell genes from C). Bar lengths correspond to normalized enrichment score (NES). Positive NES values indicate enrichment in SD + PR and negative values indicate enrichment in PD. Asterisks denote significance as defined in the figure. E UMAP of myeloid cell subclustering with cluster annotations. F Box plots displaying the frequency of myeloid cells belonging to a specific subcluster per patient comparing PD (n = 9), SD (n = 2), and PR (n = 2). Horizontal line indicates median. G Plots of DESeq2-normalized counts for highlighted genes differentially expressed by myeloid cells between categories of best response post-cryoablation (n = 13). FDR-adjusted p-values are for two-sided DESeq2 Wald test differential expression results; p_benefit denotes grouping SD + PR. H Timeline summary of patient course for patients with available pre- and post-cryoablation biopsies. I Box plots displaying the percent of immune cells in each lineage per sample, with lines connecting pre- and post-treatment samples from the same patient (n = 4). Box plots indicate interquartile range, with horizontal line indicating median, and whiskers extending to maxima and minima excluding outliers (>1.5 * IQR). PD progressive disease, SD stable disease, PR partial response. Source data for Fig are provided as a Source Data file.