Diagnosis of Leptomeningeal Metastasis in Women With Breast Cancer Through Identification of Tumor Cells in Cerebrospinal Fluid Using the CNSide™ Assay.

INTRODUCTION: Diagnosis of LM is limited by low sensitivity of cerebrospinal fluid (CSF) cytopathology. Detecting tumor cells in CSF (CSF-TCs) might be more sensitive. We evaluated if CNSide (CNSide), a novel assay for tumor cell detection in CSF, can detect CSF-TCs better than conventional CSF cytology. METHODS: We enrolled adults with metastatic breast cancer and clinical suspicion for LM to undergo lumbar puncture (LP) for CSF cytopathology and CNSide. CNSide captured CSF-TCs using a primary 10-antibody mixture, streptavidin-coated microfluidic channel, and biotinylated secondary antibodies. CSF-TCs were assessed for estrogen receptor (ER) expression by fluorescent antibody and HER2 amplification by fluorescent in situ hybridization (FISH). CSF cell-free DNA (cfDNA) was extracted for next-generation sequencing (NGS). Leptomeningeal disease was defined as positive CSF cytology and/or unequivocal MRI findings. We calculated sensitivity and specificity of CSF cytology and CNSide for the diagnosis of LM. RESULTS: Ten patients, median age 51 years (range, 37-64), underwent diagnostic LP with CSF evaluation by cytology and CNSide. CNSide had sensitivity of 100% (95% Confidence Interval [CI], 40%-100%) and specificity of 83% (95% CI, 36%-100%) for LM. Among these patients, concordance of ER and HER2 status between CSF-TCs and metastatic biopsy were 60% and 75%, respectively. NGS of CSF cfDNA identified somatic mutations in three patients, including one with PIK3CA p.H1047L in blood and CSF. CONCLUSIONS: CNSide may be a viable platform to detect CSF-TCs, with potential use as a diagnostic tool for LM in patients with metastatic breast cancer. Additional, larger studies are warranted.

LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2-cFLIP-NF-κB signaling axis.

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contributes to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrate that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T-cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

Targeting Human-Cytomegalovirus-Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoprotein B Bispecific Antibody.

The host immune response to human cytomegalovirus (HCMV) is effective against HCMV reactivation from latency, though not sufficient to clear the virus. T cells are primarily responsible for the control of viral reactivation. When the host immune system is compromised, as in transplant recipients with immunosuppression, HCMV reactivation and progressive infection can cause serious morbidity and mortality. Adoptive T cell therapy is effective for the control of HCMV infection in transplant recipients. However, it is a highly personalized therapeutic regimen and is difficult to implement in routine clinical practice. In this study, we explored a bispecific-antibody strategy to direct non-HCMV-specific T cells to recognize and exert effector functions against HCMV-infected cells. Using a knobs-into-holes strategy, we constructed a bispecific antibody in which one arm is specific for CD3 and can trigger T cell activation, while the other arm, specific for HCMV glycoprotein B (gB), recognizes and marks HCMV-infected cells based on the expression of viral gB on their surfaces. We showed that this bispecific antibody was able to redirect T cells with specificity for HCMV-infected cells in vitro In the presence of HCMV infection, the engineered antibody was able to activate T cells with no HCMV specificity for cytokine production, proliferation, and the expression of phenotype markers unique to T cell activation. These results suggested the potential of engineered bispecific antibodies, such as the construct described here, as prophylactic or therapeutic agents against HCMV reactivation and infection.