Background Tertiary lymphoid structures (TLSs) have been associated with response to immune checkpoint inhibitors (ICIs) in multiple solid tumors.1–3 TLS definitions have varied across reports and TLS identification has typically required expert pathologist assessment, introducing subjectivity and variability. Multiplex immunofluorescence (mIF) analysis can provide quantitative measures of the frequency and spatial orientation of immune cell populations, with the potential to inform standardized biomarkers and more objective reporting of TLS-like features. Using a 12-plex mIF panel, we evaluated the frequency and spatial proximity of stromal CD20+ B cells as predictive biomarkers of ICI response in melanoma, and compared their performance to that of pathologist-assessed lymphoid aggregates (PALA).
Methods Pre-treatment whole-tissue sections from 53 ICI-treated patients with unresectable melanoma were stained with a mIF panel including CD8, CD3, CD20, PD1, PD-L1, CD68, FoxP3, TCF1/7, TOX, Ki67, LAG-3, and Sox10/pancytokeratin. Cell segmentation and tissue classification were performed using HALO (Indica Labs), categorizing all cells into stromal and intratumoral compartments. Frequencies of 27 pre-specified immune phenotypes were calculated as the fraction of all cells within each compartment. Spatial proximity of phenotype pairs was calculated using a hypothesized interaction distribution (HID)4 with a pairing distance of 30 µm. A board-certified pathologist blinded to the mIF results independently scored cases for the presence of lymphoid aggregates based on hematoxylin and eosin stains. Clinical endpoints included best overall response (BOR) and progression-free survival (PFS) by RECIST v1.1.5
Results Higher stromal CD20+ frequency was associated with BOR (p = 0.00028) and longer PFS (p = 0.001) among all cases, as well as among non-lymph node metastases (n = 41; p = 0.006, p = 0.006 respectively). We identified 12 cases with both high CD20+/CD20+ HID scores and high stromal CD20+ frequency as containing digital B-cell aggregates (dBCA). dBCA presence provided greater discrimination for PFS (p = 0.0008) compared to stromal CD20+ frequency alone. There was moderate agreement between dBCA and PALA (Kappa = 0.50), and PALA positivity alone was not significantly associated with BOR or PFS. dBCA+ cases were enriched for multiple stromal T cell populations, including CD3+CD8-TCF1/7+ (p = 5.7 x 10-6), and CD3+CD8- (p = 3.92 x 10-6).
Conclusions Digital assessment of stromal B cell frequency and spatial distribution warrant further investigation as potential biomarkers of response to ICI therapy in advanced melanoma. Further characterization of T-cell populations associated with B-cell aggregates may provide additional insights into how these structures facilitate ICI-mediated anti-tumor responses.
Acknowledgements This work was supported in part by the Memorial Sloan Kettering Cancer Center (MSK) NCI Core Grant P30 CA008748.
Cabrita R, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577:561–565, doi:10.1038/s41586–019-1914–8.
Vanhersecke L, et al. Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat Cancer 2021;2:794–802, doi:10.1038/s43018–021-00232–6.
Italiano A, et al. Pembrolizumab in soft-tissue sarcomas with tertiary lymphoid structures: a phase 2 PEMBROSARC trial cohort. Nat Med 2022;28:1199–1206, doi:10.1038/s41591–022-01821–3.
Rose CJ, et al. A statistical framework for analyzing hypothesized interactions between cells imaged using multispectral microscopy and multiple immunohistochemical markers. J Pathol Inform 2013;4:S4, doi:10.4103/2153–3539.109856.
Eisenhauer EA, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228–247, doi:10.1016/j.ejca.2008.10.026.
Ethics Approval This study was approved by the Institutional Review Board of Memorial Sloan Kettering Cancer Center; approval number 19–114.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.