Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I-IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

Nuclear transfer nTreg model reveals fate-determining TCR-ß and novel peripheral nTreg precursors.

To study the development and function of "natural-arising" T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3(+)CD4(+)Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) ß-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR ß-chain was able to provide stronger TCR signals. This TCR-ß-driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3(-)CD4(+)T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells.

Tumor-localized ligation of CD3 and CD28 with systemic regulatory T-cell depletion induces potent innate and adaptive antitumor responses.

PURPOSE: Tumor-localized activation of immune cells by membrane-tethered anti-CD3 antibodies (CD3L) is under investigation to treat poorly immunogenic tumors. Here we sought to elucidate the mechanism of antitumor immunity elicited by CD3L. EXPERIMENTAL DESIGN: CD3L and CD86 were expressed on poorly immunogenic B16 melanoma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4(+) and CD8(+) T cells, natural killer T (NKT) cells, and regulatory T cells, on antitumor activity was investigated. RESULTS: B16/3L86 cells activated naïve T cells; suppressed tumor growth in subcutaneous, peritoneal, and metastasis models; and protected mice from rechallenge with B16 melanoma cells. However, in vivo antitumor activity against primary B16/3L86 tumors unexpectedly depended on NKT cells rather than CD4(+) or CD8(+) T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatory T cells unmasked latent T-cell antitumor activity; the number of activated CD8(+) T cells in tumors increased and B16/3L86 tumors were completely rejected in a CD8(+) and CD4(+) T-cell-dependent fashion. Furthermore, fibroblasts expressing CD3L and CD86 suppressed the growth of neighboring B16 cancer cells in vivo, and direct intratumoral injection of adenoviral vectors expressing CD3L and CD86 or CD3L and a membrane-tethered anti-CD28 antibody significantly suppressed the growth of subcutaneous tumors. CONCLUSIONS: Tumor-located ligation of CD3 and CD28 can activate both innate (NKT cells) and adaptive (CD4(+) and CD8(+) T cells) responses to create a tumor-destructive environment to control tumor growth, but modulation of regulatory T cells is necessary to unmask local adaptive antitumor responses.

Novel Circulating Tumor Cell Assay for Detection of Colorectal Adenomas and Cancer.

OBJECTIVES: There is a significant unmet need for a blood test with adequate sensitivity to detect colorectal cancer (CRC) and adenomas. We describe a novel circulating tumor cell (CTC) platform to capture colorectal epithelial cells associated with CRC and adenomas. METHODS: Blood was collected from 667 Taiwanese adults from 2012 to 2018 before a colonoscopy. The study population included healthy control subjects, patients with adenomas, and those with stage I-IV CRC. CTCs were isolated from the blood using the CellMax platform. The isolated cells were enumerated, and an algorithm was used to determine the likelihood of detecting adenoma or CRC. Nominal and ordinal logistic regression demonstrated that CTC counts could identify adenomas and CRC, including CRC stage. RESULTS: The CellMax test demonstrated a significant association between CTC counts and worsening disease status (Cuzick's P value < 0.0001) with respect to the adenoma-carcinoma sequence. The test showed high specificity (86%) and sensitivity across all CRC stages (95%) and adenomatous lesions (79%). The area under the curve was 0.940 and 0.868 for the detection of CRC and adenomas, respectively. DISCUSSION: The blood-based CTC platform demonstrated high sensitivity in detecting adenomas and CRC, as well as reasonable specificity in an enriched symptomatic patient population. TRANSLATIONAL IMPACT: If these results are reproduced in an average risk population, this test has the potential to prevent CRC by improving patient compliance and detecting precancerous adenomas, eventually reducing CRC mortality.

CSF-ctDNA SMSEQ Analysis to Tailor the Treatment of a Patient with Brain Metastases: A Case Report.

Brain metastases are the most common neurological complications of adult cancers, accounting for more than half of brain tumors. The incidence of brain metastases may be increasing due to improved detection of small lesions by advanced imaging technologies. Given the fast evolution of targeted and immunotherapy regimens, it is essential to serially assess brain malignancies during the disease course for disease monitoring and tailoring of the therapeutic management. For such serial and repetitive assessment, cerebrospinal fluid (CSF) could be the biological fluid of choice to supplement cytology examination for the presence or absence of CNS malignancy, as well as provide extensive information on tumor mutational profile for personalization of treatment. The case described here emphasizes the importance of CSF-ctDNA analysis with the CellMax SMSEQ technology that led to treatment adjustment resulting in clinical remission of the patient.

Liquid Biopsy Prevents Inaccurate Her2 Status Determination by in situ Hybridization in a Patient with Invasive Ductal Adenocarcinoma of the Breast: Case Report.

Utilization of circulating tumor DNA as a novel and noninvasive test for diagnosis confirmation, therapy selection, and cancer surveillance is a rapidly growing area of interest. In the wake of FDA approval of a liquid biopsy test, it is important for clinicians to acknowledge the obvious clinical utility of liquid biopsy for cancer management throughout the course of the disease. This case report describes a female with invasive ductal adenocarcinoma of the breast, where liquid biopsy was instrumental for her cancer characterization and personalized therapy selection.

The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering.

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

Autoimmune bone marrow environment severely inhibits B cell development by inducing extensive cell death and inhibiting proliferation.

The spontaneous scurfy (sf) mutation in mice results in a complete loss of Tregs, leading to a lethal, multi-system autoimmune syndrome. We have carefully examined B lymphopoiesis in sf mice. Paradoxically, the B cell numbers at all developmental stages including pro-B, pre-B, immature and mature B cells are significantly decreased in the BM and spleen of sf mice, compared to that of wild-type littermate controls. The developing B cells in sf mice exhibit profoundly elevated cell death induced by down-regulation of Bcl-XL expression and up-regulation of Fas expression. In addition, the clonal expansion of pre-B and immature B cells in sf mice is significantly reduced compared to wild-type controls. Foxp3 expression is not detectable in all stages of developing B cells in wild-type mice, indicating that the defects are B-cell extrinsic, which is further supported by the recovery of B cell maturation in BM chimeric mice. Remarkably, IFN-γ production is significantly elevated in numerous cell types in the BM of sf mice. Taken together, these results indicate that the autoimmune inflammatory marrow environment has dramatic inhibitory effects on B cell development by inducing apoptosis and suppressing proliferation of developing B cells.

A simple method for the production of recombinant proteins from mammalian cells.

Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal post-translational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINE (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5% serum. Culture medium was collected every 2-3 days and polyhistidine-tagged proteins were purified by ammonium sulphate precipitation and Ni(2+)-nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human beta-glucuronidase, mouse beta-glucuronidase, aminopeptidase N (CD13) and a single-chain antibody-enzyme fusion protein, were obtained with yields of 3-6 mg/l of culture medium. Fc-tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6-12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post-translational modifications.