Quantification of PD-L1 and PD-1 expression on tumor and immune cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry.

OBJECTIVE: We report a truly quantitative technology for PD-L1 expression in non-small cell lung cancer (NSCLC). In addition, we present a non-enzymatic technology that creates a cell suspension from fresh tumor tissue so that either fine-needle aspiration (FNA) or fresh tissue can be used in this assay. METHODS: Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, California) was performed on 4-mm punch biopsies. An FNA was taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, and PD-L1 and then stained with DAPI to identify intact, single cells, and to analyze cell cycle. RESULTS: Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r 2 - 0.8) for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1 % cutoff for positivity in the tumor cell population and a 1 % cutoff of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive. Ten of 12 lung tumor samples were concordant while 2 were discordant. PD-L1 expression by flow cytometry varied widely (1.2-89.4 %) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population. Fine, unequivocal quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue.

Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood.

Plasma viral load and CD4 counts are effective for clinical monitoring, but they do not give a full representation of HIV-1 quasispecies in cellular reservoirs, the major repository of replication-competent HIV-1 in infected individuals. We sought to develop a diagnostic system that might stimulate the replication-competent HIV-1 reservoirs for enhanced clinical monitoring, including selection of antiretroviral regimens. Whole-blood samples from 45 HIV-infected individuals were collected into 1 ViraStim HIV-1 activation tube and 1 EDTA tube. Samples were tested for viral load and cell type-specific HIV-1 replication. Further, 7 matched activated/nonactivated samples were sequenced using the Trugene HIV-1 genotyping kit. The percentage of patients with replication-competent virus in peripheral blood mononuclear cells (PBMCs) varied, depending on the baseline plasma viral load in the EDTA tubes. Six out of 24 patients with a starting plasma viral load of <20 copies/ml (cp/ml), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml, and 8 out of 13 patients with starting viral loads of >1,000 all showed increases in viral replication of >5-fold. These increases came from cellular reservoirs in blood as determined by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). When resistance genotypes in plasma from activation tubes were compared to those from EDTA tubes for 7 patients, all patients showed additional mutations in the activation tube, while 3 patients demonstrated additional genotypic resistance determinants. We show that HIV-1 viral replication can be stimulated directly from infected whole blood. The sequencing results showed that 3 of 7 cases demonstrated additional drug resistance following stimulation.

Concordance of PD-L1 Expression Detection in Non-Small Cell Lung Cancer (NSCLC) Tissue Biopsy Specimens Between OncoTect iO Lung Assay and Immunohistochemistry (IHC).

OBJECTIVES: We report on the validity of a fully quantitative technology to determine tumor cells' PD-L1 expression compared with a standard immunohistochemical (IHC) assay in non-small cell lung cancer. METHODS: Nineteen fresh tissue specimens were processed into single-cell suspensions using the IncellPREP Kit. Cells were treated with the OncoTect iO Lung Assay, which quantitatively assessed tumor-infiltrating lymphocytes (TILs), DNA content, and PD-L1 expression on diploid and aneuploid tumor populations. RESULTS: Comparison of the OncoTect iO Lung Assay with IHC revealed a concordance of 95% overall (negative percent agreement, 97%; positive percent agreement, 89%). PD-L1 expression varied depending on the DNA content. The number of TILs and antigen-presenting cells (APCs) was significantly decreased in tumor compared with normal lung tissue. CONCLUSIONS: The nonsubjective OncoTect iO Lung Assay has been shown to be at least as accurate and sensitive as IHC for the detection of PD-L1 expression while providing additional information to guide treatment.

Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells.

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.