Figure 1. TAMs are the predominant source of PD-L1 in CCA. (A) Representative images (left and middle panels) of PD-L1 (brown staining, black arrowhead) plus CD68 (red staining, red arrowhead) coimmunostaining (n = 33) and PD-L1 (brown staining) plus CK-19 (red staining) coimmunostaining (n = 18) in human resected CCA specimens. Percentage of patients with positive PD-L1/CD68 costaining and PD-L1/CK19 costaining, respectively (right panel). Scale bars: 40 μm. (B) Histograms show expression of PD-L1 + macrophages in human CCA tumors. (C-F) Flow cytometry analysis of normal WT mouse livers (from WT mice without tumors) as well as adjacent livers and tumors of mice 28 days after orthotopic implantation of 1 × 10 6 SB (murine CCA) cells. (C) Percentage of PD-L1 + macrophages (Mφ) of total macrophages (CD45 + CD11b + F4/80 + ) in WT mouse normal liver, tumor-adjacent liver, or tumor. Fluorescence Minus One (FMO) controls were used for each independent experiment to establish gates (See Supplemental Figure 1A for gating strategy) (n ≥ 8). Representative histograms show expression of PD-L1 + macrophages. (D) Percentage of CD206 + TAMs (left panel) and PD-L1 + CD206 + TAMs (middle panel) of F4/80 int macrophages (CD45 + CD11b + F4/80 int ) in WT mouse liver, tumor-adjacent liver, or tumor. Representative contour plots (right panel) show CD206 and PD-L1 expression of F4/80 int macrophages (n ≥ 7). (E) Percentage of PD-L1 + CD206macrophages or PD-L1 + CD206 + macrophages (CD11b + F4/80 + ) of CD45 + cells from SB tumors (n = 28). (F) Percentage of PD-L1 expression in myeloid cells from SB tumors.

Background and Objective Currently, computed tomography‐guided transthoracic biopsy (CTTB) is the most accurate diagnostic approach for pulmonary nodules suspected of malignancy. Traditional bronchoscopy has shown suboptimal diagnostic sensitivity, but the emergence of robotic‐assisted bronchoscopy (RAB) has the potential to improve diagnostic accuracy, maximize diagnostic yield and complete mediastinal and hilar staging in a single procedure. We aim to assess the efficacy and diagnostic performance of RAB compared to CTTB for diagnosing pulmonary nodules suspected of lung cancer. Methods A multicenter retrospective review of consecutive patients who underwent RAB and CTTB for evaluating pulmonary nodules from January 2019 to March 2021 at Mayo Clinic Florida and Mayo Clinic Rochester, United States. Clinical and demographic information, nodule characteristics, outcomes and complications were compared between RAB and CTTB. Results A total of 225 patients were included: 113 in the RAB group and 112 in the CTTB group. Overall diagnostic yield was 87.6% for RAB and 88.4% for CTTB. For malignant disease, RAB had a sensitivity of 82.1% and a specificity of 100%, CTTB had a sensitivity of 88.5% and a specificity of 100%. Complication rate was significantly higher for CTTB compared to RAB (17% vs. 4.4%; p = 0.002). Conclusion RAB, when available, can be as accurate as CTTB for sampling pulmonary nodules with similar or reduced complications and should be considered as a means for nodule biopsy, particularly when mediastinal staging is also clinically warranted.

The Hippo pathway effector Yes-associated protein (YAP) is localized to the nucleus and transcriptionally active in a number of tumor types, including a majority of human cholangiocarcinomas (CCA). YAP activity has been linked to chemotherapy resistance and has been shown to rescue KRAS and BRAF inhibition in RAS/RAF driven cancers; however the underlying mechanisms of YAP-mediated chemoresistance have yet to be elucidated. Herein, we report that the tyrosine phosphatase SHP2 directly regulates the activity of YAP by dephosphorylating pYAP Y357 even in the setting of RAS/RAF mutations, and that diminished SHP2 phosphatase activity is associated with chemoresistance in CCA. A screen for YAP interacting tyrosine phosphatases identified SHP2, and characterization of CCA cell lines demonstrated an inverse relationship between SHP2 levels and pYAP Y357 . Human sequencing data demonstrated lower SHP2 levels in CCA tumors as compared to normal liver. Cell lines with low SHP2 expression and higher levels of pYAP Y357 were resistant to gemcitabine and cisplatin. In CCA cells with high levels of SHP2, pharmacologic inhibition or genetic deletion of SHP2 increased YAP Y357 phosphorylation and expression of YAP target genes, including the antiapoptotic regulator MCL1, imparting resistance to gemcitabine and cisplatin. In vivo evaluation of chemotherapy sensitivity demonstrated significant resistance in xenografts with genetic deletion of SHP2; which could be overcome utilizing an MCL1 inhibitor.

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