Abstract GS1-00: Single-cell spatial analysis by imaging mass cytometry and immunotherapy response in triple-negative breast cancer (TNBC) in the NeoTRIPaPDL1 trial

Abstract

Background Immunecheckpoint inhibitors are effective in early and advanced TNBC, however only aminority of patients benefit making precision immune-oncology a major unmetneed. Imaging mass cytometry (IMC) enables high dimensional tissue imaging atsubcellular resolution for assessment of TNBC ecosystems, providing informationon cell type composition, functional status, and spatial organisation. Methods InNeoTRIP patients with TNBC were randomized to eight cycles ofnab-paclitaxel/carbo (CT) with/without atezolizumab (CTA). Forty-four proteinsspanning cancer cells and the tumor microenvironment (TME) were assessed onpre-treatment biopsies (n=243/280; 86.8% evaluable after QC). FFPE samples werelabelled with antibodies conjugated to isotopically pure rare earth metalreporters and profiled at one micron resolution by IMC. For each sample, wehave generated three high dimensional images that encompass the tumor,tumor-stroma interface and adjacent stroma. We investigated the association ofprotein expression assessed separately for epithelial and TME cells, cellphenotypes, and spatial architectures with PD-L1 status (Ventana SP142),stromal TILs, TNBC types and pathological complete response (pCR). 237 patients(84.6%) have both IMC and RNA-seq available allowing for comparison with genesignatures derived from HALLMARK,ConsensusTME immune cell types, and Nanostring. Results Across243 samples we identify just over one million single cells. By supervised clustering,we defined 37 robust cell phenotypes. PD-L1-positive tumors, high stromal TILsand TNBC type were characterized by extreme heterogeneity and unique cell-type andspatial TME composition. Severalbiomarkers demonstrated a significant test for interaction. Considering proteinexpression, GATA3 and CD20 on TME, HLA-DR in epithelial cells and Ki67 assessedboth in epithelial and TME, had a significant test for interaction (p <0.05). For all these biomarkers, high expression (above median) was associatedwith an increase of pCR of >10% in favour of atezolizumab, whereas lowerexpression group demonstrated a similar pCR rate among arms.Two cellphenotypes, PD-L1+IDO+ antigen presenting cells (APCs) and CD56+ neuroendocrine(NE) epithelial cells, had a significant test for interaction. Higherexpression of these biomarkers was associated with higher likelihood of pCR in CTAarm, but not in CT arm. For example, PD-L1+IDO+APCs in the CTA arm wereassociated with pCR proportions of 64.6% and 24.6% for above- and below-mediangroups respectively (OR4.5 [2.01-10.1], p<0.001).Mostof these tests of interaction retained significance after adjustment by PD-L1status and stromal TILs. Notably, none among 61 gene-expression basedimmune-related pathways and 7 proliferation-related signatures demonstrated a significant test ofinteraction. Resultsof systematic multi-tiered image analysis at the levels of cell-cellinteractions and recurrent higher order multicellular complexes defining TNBC ecosystemsidentified by graph-based methods will be presented at the meeting. Conclusions Imaging mass cytometry provides a morecomprehensive overview of TNBC heterogeneity at a single-cell level withspatial resolution. Bulk protein or gene expression might deliver limitedpredictive information because it does not consider the cell compartment ofexpression. Precise cell phenotyping highlights the predictive role ofPD-L1+IDO+APCs and CD56+NE epithelial cells. Overall, we demonstrated that IMCis feasible in a large, randomized trial and provides independent predictiveinformation on immune checkpoint inhibitors benefit to PD-L1, TILs and gene-expressionprofiles. Citation Format: Giampaolo Bianchini, Xiao Qian Wang, Esther Danenberg, Chiun-Sheng Huang, Daniel Egle, Maurizio Callari, Begoña Bermejo, Claudio Zamagni, Marc Thill, Anton Anton, Matteo Dugo, Stefania Zambelli, Stefania Russo, Eva Maria Ciruelos, Richard Greil, Vladimir Semiglazov, Marco Colleoni, Catherine Kelly, Gabriella Mariani, Lucia Del Mastro, Balázs Győrffy, Olivia Biasi, Pinuccia Valagussa, Giuseppe Viale, Luca Gianni, H Raza Ali. Single-cell spatial analysis by imaging mass cytometry and immunotherapy response in triple-negative breast cancer (TNBC) in the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-00.

Update Breast Cancer 2022 Part 1 – Early Stage Breast Cancer

“…A total of 43 markers that can differentiate between immune cells and epithelial cells were assessed using an antibody- and mass spectroscopy-based system 63 in 237 of the 280 patients included. Indeed, two patterns could be identified that predicted a high pCR rate (approximately 65%) and a low pCR rate (approximately 25 – 35%) in patients treated with atezolizumab, whereas no predictive value was demonstrated for the spatial pattern in patients treated with chemotherapy alone 62 .…”

“…Im Rahmen der NeoTRIP-Studie, die eine Therapie mit Carboplatin und nab-Paclitaxel mit einer Therapie mit Carboplatin, nab-Paclitaxel und Atezolizumab verglich, wurden nun solche Analysen durchgeführt 62 . Insgesamt 43 Marker, die zwischen Immunzellen und epithelialen Zellen unterscheiden können, wurden mittels eines auf Antikörpern und Massenspektroskopie beruhenden Systems 63 bei 237 der 280 eingeschlossenen Patientinnen untersucht.…”

“…65%) und eine niedrige pCR-Rate (ca. 25 – 35%) vorhersagen konnten, während bei Patientinnen, die nur mit Chemotherapie behandelt worden waren, kein prädiktiver Wert des spatialen Musters nachgewiesen werden konnte 62 .…”

“…Such analyses have now been performed as part of the NeoTRIP trial, which compared a regimen of carboplatin and nab-paclitaxel with a regimen of carboplatin, nab-paclitaxel, and atezolizumab 62 . A total of 43 markers that can differentiate between immune cells and epithelial cells were assessed using an antibody- and mass spectroscopy-based system 63 in 237 of the 280 patients included.…”

Dissecting Tumor-Immune Microenvironment in Breast Cancer at a Spatial and Multiplex Resolution

“…Moreover, baseline stromal LAG3 expression and altered densities of CD4+PD1+ T cells after three weeks of chemotherapy were favorable prognostic factors [61,76]. To date, the largest application of imaging mass cytometry has been applied in 243 TNBC of NeoTRIPaPDL1 phase III trial, where high densities of PDL1+IDO+ antigen presenting cells and spatial correlation of cancer cells with T cell subsets (CD8+PD1+, CD8+GZMB+) and B cells (CD20+) accounted for higher pCR rates in atezolizumab arm, independent of sTILs and PD-L1 IHC [91]. In summary, multiplex in situ evaluation of immune cells' densities, spatial distribution and functional status may provide the necessary step forward in identification of TIME-related biomarkers (Table 3).…”