Multimodality analysis confers a prognostic benefit of a T-cell infiltrated tumor microenvironment and peripheral immune status in patients with melanoma

Abstract

Background: We previously reported results from a phase 1 study testing intratumoral recombinant poliovirus, lerapolturev, in 12 melanoma patients. All 12 patients received anti-PD-1 systemic therapy before lerapolturev, and 11 of these 12 patients also received anti-PD-1 after lerapolturev. In preclinical models lerapolturev induces intratumoral innate inflammation that engages antitumor T cells. In the current study, prelerapolturev and postlerapolturev tumor biopsies and blood were evaluated for biomarkers of response.

Methods: The following analyses were performed on tumor tissue (n=11): (1) flow cytometric assessment of immune cell density, (2) NanoString Digital Spatial profiling of protein and the transcriptome, and (3) bulk RNA sequencing. Immune cell phenotypes and responsiveness to in vitro stimulation, including in vitro lerapolturev challenge, were measured in peripheral blood (n=12).

Results: Three patients who received anti-PD-1 therapy within 30 days of lerapolturev have a current median progression-free survival (PFS) of 2.3 years and had higher CD8+T cell infiltrates in prelerapolturev tumor biopsies relative to that of 7 patients with median PFS of 1.6 months and lower CD8+T cell infiltrates in prelerapolturev tumor biopsies. In peripheral blood, four patients with PFS 2.3 years (including three that received anti-PD-1 therapy within 30 days before lerapolturev and had higher pretreatment tumor CD8+T cell infiltrates) had significantly higher effector memory (CD8+, CCR7-, CD45RA-) but lower CD8+PD-1+ and CD4+PD-1+ cells compared with eight patients with median PFS 1.6 months. In addition, pretreatment blood from the four patients with median PFS 2.3 years had more potent antiviral responses to in vitro lerapolturev challenge compared with eight patients with median PFS 1.6 months.

Conclusion: An inflamed pretreatment tumor microenvironment, possibly induced by prior anti-PD-1 therapy and a proficient peripheral blood pretreatment innate immune response (antiviral/interferon signaling) to lerapolturev was associated with long term PFS after intratumoral lerapolturev in a small cohort of patients. These findings imply a link between intratumoral T cell inflammation and peripheral immune function.

Trial registration number: NCT03712358.

Keywords: immunity, innate; oncolytic virotherapy; tumor microenvironment.

Conflict of interest statement

Competing interests: GMB reports clinical trial funding from Istari Oncology, Delcath, Oncosec Medical, Replimune, and Checkmate Pharmaceuticals paid to Duke University. AnS receives research funding paid to the institution from Bristol Myers Squibb, Immunocore, Merck, Pfizer Advisory roles; and has served on advisory boards for Array, Novartis, Iovance, and Regeneron. MCB, DDB and MG are paid consultants of Istari Oncology, Inc. and MG and DDB are scientific advisors for Istari Oncology; DDB and MG hold equity in Istari Oncology. MCB, DDB, MG and SKN own intellectual property related to this research, which has been licensed to Istari Oncology. Duke University (Licensor of PVSRIPO) has a financial interest in Istari Oncology. All other authors declare they have no competing interests.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Flow cytometry and RNA sequencing analysis of tumor tissue. (A) Number of CD8+T cells and (B) number of CD4+T cells in tumor tissue as assessed by flow cytometry of fresh tumor tissue. (C)- Number of CD8+T cells (left) and CD4+T cells (right) in pretreatment tumor tissue. (D) CIBERSORT predicted (bulk RNA-sequencing) enrichment of CD8+T cells and active memory CD4+T cells in pretreatment tumor tissue Please see online supplemental figure S1 for full heatmap of CIBERSORT enrichment scores for each patient. Patients in red have current median PFS 2.3 years compared with patients in black text with median PFS 1.6 months. PFS, progression-free survival.

Figure 2

Analysis of tumor tissue sections. NanoString Digital Spatial Profiling (DSP) low power fluorescent images of pre-treatment tumor tissue; CD3=red, CD11c=yellow, CD163=green, DNA=blue. All images are ‘low power’= 2 mm field of view, 100 x total magnification. Number in left lower corner indicates patient number. Top row, patients with median PFS 1.6 months after lerapolturev. Bottom row, patients with median PFS 2.3 years after lerapolturev. The number of CD3+T cells as assessed by flow cytometry is also shown for each tumor. Below the DSP images, a corresponding H&E stain is also shown. Note for patient 11, the H&E demonstrates near 100% necrotic tissue in the pretreatment biopsy, as such this was dropped from analysis. PFS, progression-free survival.

Figure 3

NanoString Digital Spatial Profiling (DSP) of tumor tissue. Top left and middle panel, ‘low power’ = 2 mm field of view, 100 x total magnification, ‘high power’ = 45 μm field of view, 400 × total magnification. (A, B) Patient 2 prelerapolturev and (C, D) post-lerapolturev tumor. (A, C) Corresponding H&E. (B) DSP low power, fluorescent image, CD3=red, CD11c=yellow, CD163=green, DNA=blue. Same fluorescent markers also present in (D, F, H). B1—High power view of region 1 circled (white circle) in image B. (B2) High power view of region 2 circled (white circle) in image B. (D) DSP image low power, tumor cells can be identified as cells with irregular blue nucleus. D1—High power view of region 1 circled (white circle) in image D. D2—High power view of region 2 circled (white circle) in image D. Bottom left and middle (E, F) Patient 8 prelerapolturev and G, H postlerapolturev tumor. (E, G) Corresponding H&E stains. (F) DSP image low power. (F1) High power view of region 1 circled (white circle) in image F. (F2) High power view of region 2 circled (white circle) in image F. Tumor cells can be identified as cells with irregular blue nucleus depicted in F2, red circle. H- DSP image low power. (H1) High power view of region 1 circled (white circle) in image H. (H2) High power view of region 2 circled (white circle) in image (H. I, J) Heatmaps of selected protein expression obtained from tumor regions of interest in (I) pretreatment tumor tissue and J- CD3+compartment only. Patient identification on the y axis (right), pre-NON is pretreatment tissue collected without lerapolturev injection.

Figure 4

NanoString Digital Spatial Profiling (DSP) of tumor tissue. NanoString DSP images after fluorescent staining on pretreatment tissue, CD8=teal, CD4=red, CD11c=yellow, and DNA=blue. ‘Low power’ = 2 mm field of view, 100 × total magnification. Rectangles indicate ROIs selected based on enrichment for cell type or tumor. (A) Low power image patient 12. (B) Low power image patient 7. (C) Volcano plot comparing ROI on CD8+ cells only between patients 8, 9, 12, 5 and 7. Differentially expressed genes appear in blue. ROI, regions of interest.

Figure 5

Flow cytometry analysis of immune subsets in peripheral blood. PBMCs were analyzed for T cell memory populations and PD1 expression on CD8+ and CD4+ conventional T cells as shown at the pretreatment time point. Patients in red have median PFS of 2.3 years after lerapolturev and patients in black have median PFS 1.6 months after lerapolturev. Data brackets indicate mean ±SEM; p values are from unpaired t-test. PBMCs, peripheral blood mononuclear cells; PFS, progression-free survival.

Figure 6

Assessment of baseline peripheral immune cell function in response to lerapolturev. PBMCs from the pretreatment time point were challenged with laboratory grade PVSRIPO in vitro for 24 hours. Supernatant was tested for proinflammatory cytokines. Patients in red have median PFS of 2.3 years after lerapolturev and patients in black have median PFS 1.6 months after lerapolturev. Data brackets indicate mean ±SEM; p values are from unpaired t-test. See online supplemental figure S3 for complete results of cytokines and stimuli tested; online supplemental figure S4 presents baseline raw MFI and concentration of cytokines for all stimuli. GMCSF, granulocyte-macrophage colony-stimulating factor; MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells; PFS, progression-free survival.