Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch® System. We tested an epitope-independent method (ParsortixTM system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45−, pankeratins (K)+ cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1+CTCs, 47% had PD-L1+ and PD-L1−CTCs, and only 7% displayed exclusively PD-L1−CTCs. The percentage of PD-L1+CTCs did not correlate with the percentage of PD-L1+ in biopsies determined by immunohistochemistry (p = 0.179). Upon disease progression, all patients showed an increase in PD-L1+CTCs, while no change or a decrease in PD-L1+CTCs was observed in responding patients (n = 11; p = 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1+CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.

Mast cells are haematopoietic cells that arise from pluripotent precursors of the bone marrow. They play immunomodulatory roles in both health and disease. When appropriately activated, mast cells undergo degranulation, and preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on various inflammatory settings are closely associated with the enzymatic characteristics of tryptase, the main granule compound of mast cells. Tryptase degranulation is often linked with the development of an immune response, allergy, inflammation, and remodelling of tissue architecture. Tryptase also represents an informative diagnostic marker of certain diseases and a prospective target for pharmacotherapy. In this review, we discuss the current knowledge about mast cell tryptase as one of the mast cell secretome proteases. The main points of the reviewed publications are highlighted with our microscopic images of mast cell tryptases visualized using immunohistochemical staining.

INTRODUCTION Tumor mutational burden (TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome. Immunotherapy benefit has been associated with TMB assessed by whole exome sequencing (wesTMB) and by gene panel sequencing (psTMB). The initiatives of Quality in Pathology (QuIP) and Friends of Cancer Research (FoCR) have jointly addressed the need for harmonization between TMB testing options in tissues. This QuIP study identifies critical sources of variation in psTMB assessment. METHODS Twenty samples from three tumor types (LUAD, HNSC, COAD) with available WES data were analyzed for psTMB, using six panels across 15 testing centers. Inter-laboratory and inter-platform variation including agreement on variant calling and TMB classification were investigated. Bridging factors to transform psTMB to wesTMB values were empirically derived. The impact of germline filtering was evaluated. RESULTS Sixteen samples demonstrated low interlaboratory and interpanel psTMB variation with 87.7% of pairwise comparisons showing a Spearman's >0.6. A wesTMB cutpoint of 199 missense mutations projected to psTMB cutpoints between 7.8 and 12.6 muts/Mbp; the corresponding psTMB and wesTMB classifications agreed in 74.9% of cases. For three-tier classification with cutpoints of 100 and 300 mutations, agreement was observed in 76.7%, weak misclassification in 21.8%, and strong misclassification in 1.5% of cases. Confounders of psTMB estimation included fixation artifacts, DNA input, sequencing depth, genome coverage, and variant allele frequency cutpoints. CONCLUSIONS This study provides real-world evidence that all evaluated panels can be used to estimate TMB in a routine diagnostic setting and identifies important parameters for reliable tissue TMB assessment that require careful control. As complex/composite biomarkers beyond TMB are likely playing an increasing role in therapy prediction, the efforts by QuIP and FoCR also delineate a general framework and blueprint for the evaluation of such assays.

Nitrogen‐containing bisphosphonates (N‐BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N‐BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA‐6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA‐6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP‐ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N‐BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.

The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated.