Artificial Intelligence-Driven Morphology-Based Enrichment of Malignant Cells from Body Fluid.

Cell morphology is a fundamental feature used to evaluate patient specimens in pathologic analysis. However, traditional cytopathology analysis of patient effusion samples is limited by low tumor cell abundance coupled with the high background of nonmalignant cells, restricting the ability of downstream molecular and functional analyses to identify actionable therapeutic targets. We applied the Deepcell platform that combines microfluidic sorting, brightfield imaging, and real-time deep learning interpretations based on multidimensional morphology to enrich carcinoma cells from malignant effusions without cell staining or labels. Carcinoma cell enrichment was validated with whole genome sequencing and targeted mutation analysis, which showed a higher sensitivity for detection of tumor fractions and critical somatic variant mutations that were initially at low levels or undetectable in presort patient samples. Our study demonstrates the feasibility and added value of supplementing traditional morphology-based cytology with deep learning, multidimensional morphology analysis, and microfluidic sorting.

Comparison of Afirma Gene Expression Classifier with Gene Sequencing Classifier in indeterminate thyroid nodules: A single-institutional experience.

INTRODUCTION: The Afirma test has been used in the diagnosis of cytologically indeterminate thyroid nodules to reduce diagnostic uncertainty and unnecessary surgeries. Gene Sequencing Classifier (GSC) was developed to improve the positive predictive value and overall test performance of Gene Expression Classifier (GEC). Here we present our experience comparing the performance of first-generation assay of Afirma (GEC) with the new assay (GSC). METHODS: Retrospective analysis was performed on all Bethesda III and IV cytology thyroid nodules tested with GEC and GSC. Test performance was evaluated by surgical pathology outcomes. RESULTS: In total, 167 cases were tested with GEC, of which 49% were reported as benign. Fourteen cases had surgical follow-up with 11 benign, one non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and two malignant diagnoses. Of the 167 cases, 51% had suspicious GEC result. Fifty-seven of these suspicious GEC cases had surgical follow-up with 28 benign, nine NIFTP and 20 malignant histology. There 133 cases tested with GSC, of which 61% were reported as benign. Ten cases had surgical follow-up, all of which showed benign results and 32% of the cases were tested as suspicious. Thirty-six cases with suspicious GSC had surgical follow-up. Fourteen of them had benign, five NIFTP, and 17 malignant surgical pathology. Based on molecular testing, surgical resection could have been be prevented 61% with GSC, compared to 49% with GEC test. CONCLUSION: Our experience shows that GSC has a better test performance than GEC. Also, our data support that GSC identify more cases as benign and reduces the number of unnecessary surgeries compared to GEC.

Identification of novel PIEZO1 variants using prenatal exome sequencing and correlation to ultrasound and autopsy findings of recurrent hydrops fetalis.

Nonimmune hydrops fetalis (NIHF) is a rare disorder with a high perinatal mortality of at least 50%. One cause of NIHF is generalized lymphatic dysplasia (GLD), a rare form of primary lymphedema of the extremities and systemic involvement including chylothoraces and pericardial effusions. An autosomal recessive form of GLD has been described, caused by variants in the PIEZO1 gene. It has been reported clinically to cause NIHF and childhood onset of facial and limb lymphedema, most of which were diagnosed postnatally. We present a case of a woman with recurrent pregnancies affected by NIHF because of novel compound heterozygous variants in the PIEZO1 gene diagnosed prenatally using exome sequencing (ES). Two variants in PIEZO1 (c.3206G>A and c.6208A>C) were identified that were inherited from the father and mother, and are predicted to cause a nonsense and missense change, respectively, in the PIEZO1 subunits. Ultrasound demonstrated severe bilateral pleural effusions, whole body edema and polyhydramnios. Histopathology revealed an increased number of lymphatic channels, many of which showed failure of luminal canalization. Sanger sequencing confirmed the same variants in a prior fetal demise. We provide phenotypic correlation with ultrasound and autopsy finding, review PIEZO1 variants as a cause of GLD and discuss the uses of prenatal ES to date.

The dimeric structure of factor XI and zymogen activation.

Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit.

A sequential mechanism for exosite-mediated factor IX activation by factor XIa.

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg(145) to form fIXα, and then after Arg(180) to form the protease fIXaß. FIXα is released from fXIa, and must rebind for cleavage after Arg(180) to occur. Catalytic efficiency of cleavage after Arg(180) is 7-fold greater than for cleavage after Arg(145), limiting fIXα accumulation. FXIa contains four apple domains (A1-A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases K(m) for fIX cleavage after Arg(145) and Arg(180) 25- and ≥ 90-fold, respectively, and markedly decreases k(cat) for cleavage after Arg(180). Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca(2+). Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg(180) than for cleavage after Arg(145), resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca(2+)-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain.

The mechanism underlying activation of factor IX by factor XIa.

Factor XI (fXI) is the zymogen of a plasma protease, factor XIa (fXIa), that contributes to thrombin generation during blood coagulation by proteolytic conversion of factor IX (fIX) to factor IXaß (fIXaß). There is considerable interest in fXIa as a therapeutic target because it contributes to thrombosis, while serving a relatively minor role in hemostasis. FXI/XIa has a distinctly different structure than other plasma coagulation proteases. Specifically, the protein lacks a phospholipid-binding Gla-domain, and is a homodimer. Each subunit of a fXIa dimer contains four apple domains (A1 to A4) and one trypsin-like catalytic domain. The A3 domain contains a binding site (exosite) that largely determines affinity and specificity for the substrate fIX. After binding to fXIa, fIX undergoes a single cleavage to form the intermediate fIXα. FIXα then rebinds to the A3 domain to undergo a second cleavage, generating fIXaß. The catalytic efficiency for the second cleavage is ~7-fold greater than that of the first cleavage, limiting fIXα accumulation. Residues at the N-terminus and C-terminus of the fXIa A3 domain likely form the fIX binding site. The dimeric conformation of fXIa is not required for normal fIX activation in solution. However, monomeric forms of fXI do not reconstitute fXI-deficient mice in arterial thrombosis models, indicating the dimer is required for normal function in vivo. FXI must be a dimer to be activated normal by the protease fXIIa. It is also possible that the dimeric structure is an adaptation that allows fXI/XIa to bind to a surface through one subunit, while binding to its substrate fIX through the other.

Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.

The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.