PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry.

OBJECTIVES: Pathologists encounter several challenges with programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) tests in malignant effusions, including lineage specification (distinction between carcinoma vs. immune and mesothelial cells), background staining, sample fixation issues and inter-observer variability. We explored flow cytometric (FC) quantification of PD-L1 expression in malignant pleural effusions of lung adenocarcinoma patients as an alternative, automated, and objective quantification method compared to PD-L1 IHC. MATERIALS AND METHODS: We examined 23 malignant pleural effusions of TTF-1-positive adenocarcinoma were subjected to FC with a panel of antibodies against CD45, CD3, CD200, EpCAM, D2-40 (podoplanin), and PD-L1 (clone MIH1). The PD-L1 gate was established using fluorescence-minus-one (FMO) isotype controls. Lineage-specific PD-L1 surface expression was quantified and the FC tumor proportion score (TPS) was assessed. PD-L1 IHC was performed on cell block sections using Dako PD-L1 IHC 22C3 pharmDx assay and assessed by two cytopathologists blinded to the FC PD-L1 TPS. RESULTS: FC analysis allowed for the distinction between carcinoma cells (CD45-/EpCAM+/D2-40-), leukocytes (CD45+/EpCAM-/D2-40-) and mesothelial cells (CD45-/EpCAM-/D2-40+). FC PD-L1 TPS ranged from 0% to 77 %, while the 22C3 IHC PD-L1 TPS ranged from 0% to 97 %. The FC and IHC TPS values correlated positively (R = 0.8). Best concordance was observed when FC was performed and cell blocks were generated in parallel (R = 0.99). FC also allowed for simultaneous PD-L1 quantification in mesothelial and T-cells. PD-L1 expression on mesothelial cells ranged from 0% to 90.9 %, which also correlated positively with IHC TPS (R = 0.54). PD-L1 expression on T-cells was limited (0.1-2.9 %). CONCLUSION: FC permits rapid, objective and lineage-specific PD-L1 surface expression quantification with limited specimen manipulation. The FC and IHC concordance was impacted by different antibody clones being used, but the positive correlation suggests potential clinical utility, especially in malignant effusion specimens.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms.

BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. METHODS: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. RESULTS/CONCLUSIONS: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 = 1) and monocytes (R2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

Phase I combination of sorafenib and erlotinib therapy in solid tumors: safety, pharmacokinetic, and pharmacodynamic evaluation from an expansion cohort.

The aims of this study were to further define the safety of sorafenib and erlotinib, given at their full approved monotherapy doses, and to correlate pharmacokinetic and pharmacodynamic markers with clinical outcome. In addition, a novel pharmacodynamic marker based on the real-time measurement of RAF signal transduction capacity (STC) is described. Sorafenib was administered alone for a 1-week run-in period, and then both drugs were given together continuously. RAF STC was assessed in peripheral blood monocytes prior to erlotinib initiation. Epidermal growth factor receptor (EGFR) expression and K-RAS mutations were measured in archival tumor samples. Changes in pERK and CD31 were determined in fresh tumor biopsies obtained pretreatment, prior to erlotinib dosing, and during the administration of both drugs. In addition, positron emission tomography-computed tomography scans and pharmacokinetic assessments were done. Eleven patients received a total of 57 cycles (median, 5; range, 1-10). Only four patients received full doses of both drugs for the entire study course, with elevation of liver enzymes being the main reason for dose reductions and delays. Among 10 patients evaluable for response, 8 experienced tumor stabilization of >or=4 cycles. Pharmacokinetic analysis revealed no significant interaction of erlotinib with sorafenib. Sorafenib-induced decrease in RAF-STC showed statistically significant correlation with time-to-progression in seven patients. Other pharmacodynamic markers did not correlate with clinical outcome. This drug combination resulted in promising clinical activity in solid tumor patients although significant toxicity warrants close monitoring. RAF-STC deserves further study as a predictive marker for sorafenib.

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers.

BACKGROUND AIMS: Enumeration of viable CD34(+) cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34(+) cells/microL blood predicts the collection of at least 0.5x10(6) CD34(+) cells/kg patient weight. From the apheresis product, infusion of 2.5x10(6) viable CD34(+) cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/microL) by day 12-14 post-infusion. METHODS: We compared the CD34(+) cell numbers derived from Flow Count-based Stem-Kit; (Beckman Coulter) and Trucount tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur and BD FACSCanto cytometers on 12 granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples. RESULTS: Comparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/microL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R(2)>0.98. CONCLUSIONS: The two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.

Quantitation of CD36 (platelet glycoprotein IV) expression on platelets and monocytes by flow cytometry: application to the study of Plasmodium falciparum malaria.

BACKGROUND: The expression of CD36 (platelet glycoprotein IV) is variable among different individuals and cannot be determined by gene analysis. Previous studies suggest that CD36 expression plays a central role in the pathophysiology of Plasmodium falciparum malaria, a disease of global significance. METHODS: We developed a flow cytometric method to quantitatively measure CD36 on monocytes and platelets from whole blood using antibodies to CD36, CD14, and CD61 directly conjugated to different fluorochromes. Commercially available fluorescent beads were used to quantify CD36 expression. RESULTS: The assay was successfully run at three different centers. African-Americans (n = 57), nonAfrican-Americans (n = 33), individuals with and without hemoglobin S (n = 15 and n = 12), and children with P falciparum malaria (n = 97) were tested. Platelet-monocyte aggregates, present to varying degrees in different anticoagulants, were eliminated from final analysis. The median fluorescence intensity (MFI) of CD36 among different subjects followed a log-normal distribution. Among African-Americans, 5% were CD36-deficient (logMFI < 1.5; MFI < 32). Expression of platelet CD36 paralleled monocyte CD36. CONCLUSIONS: Flow cytometry can be used to quantify the expression of CD36 of platelets and monocytes in EDTA whole blood. The assay will allow investigation of the relationship between CD36 and clinical outcome in malaria and other disease states.