Antiretroviral therapy effectively suppresses, but does not eradicate HIV-1 infection. Persistent low-level HIV-1 can still be detected in plasma and cellular reservoirs even after years of effective therapy, and cessation of current treatments invariably results in resumption of viral replication. Efforts to eradicate persistent HIV-1 require a comprehensive examination of the quantity and genetic composition of HIV-1 within the plasma and infected cells located in the peripheral blood and tissues throughout the body. Single-molecule techniques, such as the single-copy assay and single-genome/proviral sequencing assays, have been employed to further our understanding of the source and viral dynamics of persistent HIV-1 during long-term effective therapy. The application of the single-copy assay, which quantifies plasma HIV-1 RNA down to a single copy, has revealed that viremia persists in the plasma and CSF after years of effective therapy. This low-level HIV-1 RNA also persists in the plasma following treatment intensification, treatment with latency reversing agents, cancer-related therapy, and bone marrow transplantation. Single-genome/proviral sequencing assays genetically characterise HIV-1 populations after passing through different selective pressures related to cell type, tissue type, compartment, or therapy. The application of these assays has revealed that the intracellular HIV-1 reservoir is stable and mainly located in CD4+ memory T cells. Moreover, this intracellular HIV-1 reservoir is primarily maintained by cellular proliferation due to homeostasis and antigenic stimulation, although cryptic replication may take place in anatomic sites where treatment is sub-optimal. The employment of single-genome/proviral sequencing showed that latency reversing agents broadly activate quiescent proviruses but do not clear the intracellular reservoir. Recently, full-length individual proviral sequencing assays have been developed and the application of these assays has revealed that the majority of intracellular HIV-1 DNA is genetically defective. In addition, the employment of these assays has shown that genetically intact proviruses are unequally distributed in memory T cell subsets during antiretroviral therapy. The application of single-molecule assays has enhanced the understanding of the source and dynamics of persistent HIV-1 in the plasma and cells of HIV-infected individuals. Future studies of the persistent HIV-1 reservoir and new treatment strategies to eradicate persistent virus will benefit from the utilization of these assays.

The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir.

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.