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Companion diagnostic immunohistochemistry (IHC) tests are developed and performed without incorporating the tools and principles of laboratory metrology. Basic analytic assay parameters such as lower limit of detection (LOD) and dynamic range are unknown to both assay developers and end users. We solved this problem by developing completely new tools for IHC-calibrators with units of measure traceable to National Institute of Standards & Technology (NIST) Standard Reference Material (SRM) 1934. In this study, we demonstrate the clinical impact and opportunity for incorporating these changes into PD-L1 testing. Forty-one laboratories in North America and Europe were surveyed with newly-developed PD-L1 calibrators. The survey sampled a broad representation of commercial and laboratory-developed tests (LDTs). Using the PD-L1 calibrators, we quantified analytic test parameters that were previously only inferred indirectly after large clinical studies. The data show that the four FDA-cleared PD-L1 assays represent three different levels of analytic sensitivity. The new analytic sensitivity data explain why some patients' tissue samples were positive by one assay and negative by another. The outcome depends on the assay's lower LOD. Also, why previous attempts to harmonize certain PD-L1 assays were unsuccessful; the assays' dynamic ranges were too disparate and did not overlap. PD-L1 assay calibration also clarifies the exact performance characteristics of LDTs relative to FDA-cleared commercial assays. Some LDTs' analytic response curves are indistinguishable from their predicate FDA-cleared assay. IHC assay calibration represents an important transition for companion diagnostic testing. The new tools will improve patient treatment stratification, test harmonization, and foster accuracy as tests transition from clinical trials to broad clinical use.

Background Immunohistochemical staining of programmed death‐ligand 1 (PD‐L1) is used to determine which patients with non–small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD‐L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD‐L1 immunoreactivity was studied. Methods Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine‐needle aspiration on pneumonectomy/lobectomy specimens. Formalin‐fixed, agar‐based or CytoLyt/PreservCyt‐fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory‐developed test) antibodies. PD‐L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD‐L1–expressing human cell lines fixed in various fixatives at increasing increments in fixation duration. Results Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair‐to‐moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol‐based fixatives showed less PD‐L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory‐developed test and the 22C3 pharmDx assay. Conclusions Performing PD‐L1 staining on cytologic specimens fixed in alcohol‐based fixatives could result in false‐negative immunostaining results, whereas fixation in formalin leads to higher and more histology‐concordant PD‐L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin., Fixation of cytologic specimens in alcohol‐based fixatives results in negative effects on programmed death‐ligand 1 (PD‐L1) immunostaining when using PD‐L1 immunohistochemistry protocols validated on formalin‐fixed, paraffin‐embedded material. The use of formalin results in more histology‐concordant PD‐L1 immunostaining, and the deleterious effect of alcohol fixation potentially may be reversed to some degree by postfixation in formalin.

Selection of non-small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance. ispartof: JOURNAL OF MOLECULAR DIAGNOSTICS vol:22 issue:12 pages:1438-1452 ispartof: location:United States status: published

OBJECTIVES: Programmed death-ligand 1 (PD-L1) is the only approved predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). However, predictive PD-L1 immunohistochemistry is subject to interobserver variability. We hypothesized that a pathologist's personality influences the interobserver variability and diagnostic accuracy of PD-L1 immunoscoring.MATERIALS AND METHODS: Seventeen pathologists performed PD-L1 immunoscoring on 50 resected NSCLC tumors in three categories (RESULTS: The overall agreement among pathologists for a series of 47 tumors was substantial (kappa = 0.63). Of these, 23/47 (49%) tumors were entirely negative or largely positive, resulting in a kappa value of 0.93. The remaining 24/47 (51%) tumors had a PD-L1 score around the cutoff value, generating a kappa value of 0.32. Pathologists with high scores for conscientiousness (careful, diligent) had the least interobserver variability (r = 0.6, p = 0.009). Also, they showed a trend towards higher sensitivity (74% vs. 68%, p = 0.4), specificity (86% vs. 82%, p = 0.3) and percent agreement (83% vs. 79%, p = 0.3), although not significant. In contrast, pathologists with high scores for neuroticism (sensitive, anxious) had significantly lower specificity (80% vs. 87%, p = 0.03) and percent agreement (78% vs. 85%, p = 0.03). Also, a trend towards high interobserver variability (r = -0.3, p = 0.2) and lower sensitivity (68% vs. 74%, p = 0.3) was observed, although not significant. Pathologists with relatively high scores for conscientiousness scored fewer tumors PD-L1 positive at the ≥ 1% cut-off (r = -0.5, p = 0.03). In contrast, pathologists with relatively high scores for neuroticism score more tumors PD-L1 positive at ≥ 1% (r = 0.6, p = 0.017) and ≥ 50% cut-offs (r = 0.6, p = 0.009).CONCLUSIONS: This study is the first to demonstrate the impact of a pathologist's personality on the interobserver variability and diagnostic accuracy of immunostaining, in the context of PD-L1 in NSCLC. Larger studies are needed for validation of these findings.

Companion diagnostic immunohistochemistry (IHC) tests are developed and performed without incorporating the tools and principles of laboratory metrology. Basic analytic assay parameters such as lower limit of detection (LOD) and dynamic range are unknown to both assay developers and end users. We solved this problem by developing completely new tools for IHC-calibrators with units of measure traceable to National Institute of StandardsTechnology (NIST) Standard Reference Material (SRM) 1934. In this study, we demonstrate the clinical impact and opportunity for incorporating these changes into PD-L1 testing. Forty-one laboratories in North America and Europe were surveyed with newly-developed PD-L1 calibrators. The survey sampled a broad representation of commercial and laboratory-developed tests (LDTs). Using the PD-L1 calibrators, we quantified analytic test parameters that were previously only inferred indirectly after large clinical studies. The data show that the four FDA-cleared PD-L1 assays represent three different levels of analytic sensitivity. The new analytic sensitivity data explain why some patients' tissue samples were positive by one assay and negative by another. The outcome depends on the assay's lower LOD. Also, why previous attempts to harmonize certain PD-L1 assays were unsuccessful; the assays' dynamic ranges were too disparate and did not overlap. PD-L1 assay calibration also clarifies the exact performance characteristics of LDTs relative to FDA-cleared commercial assays. Some LDTs' analytic response curves are indistinguishable from their predicate FDA-cleared assay. IHC assay calibration represents an important transition for companion diagnostic testing. The new tools will improve patient treatment stratification, test harmonization, and foster accuracy as tests transition from clinical trials to broad clinical use.

AIM Integration of endomyocardial biopsy (EMB) in the diagnostic workup of cardiac sarcoidosis (CS) is under-recognized in current clinical practice, since capturing focal granulomas is challenging. Our aim was to describe our experience with electro-anatomic mapping (EAM)-guided EMB and provide a comprehensive review of the literature. METHODS AND RESULTS Five patients (age 49.4 ± 11.4) with suspected CS underwent EAM-guided EMB in Isala Heart Center (Zwolle, the Netherlands) between 2017 and 2019. In all patients, a 3D bipolar voltage map (

AimsInvestigate the impact of interlaboratory- and interobserver variability of immunohistochemistry on the assessment of programmed death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC).MethodsTwo tissue microarrays (TMAs) were constructed from 50 (TMA-A) and 51 (TMA-B) resected NSCLC cases, and distributed among eight centres. Immunostaining for PD-L1 was performed using Agilent’s 22C3 pharmDx Assay (pharmDx) and/or a 22C3 laboratory developed test (LDT). The interlaboratory variability of staining- and interobserver variability of scoring for PD-L1 were assessed in selected critical samples (samples at the cut-off of positivity) and non-critical samples. Also, PD-L1 epitope deterioration in time in stored unstained slides was analysed. Krippendorff’s alpha values (0=maximal, 1=no variability) were calculated as measure for variability.ResultsFor interlaboratory variability of immunostaining, the percentage of PD-L1 positive cases among centres ranged 40%–51% (1% cut-off) and 23%–30% (50% cut-off). Alpha values at 1% cut-off were 0.88 (pharmDx) and 0.87 (LDT) and at 50% cut-off 0.82 (pharmDx) and 0.95 (LDT). Interobserver variability of scoring resulted in PD-L1 positive cases ranging 29%–55% (1% cut-off) and 14%–30% (50% cut-off) among pathologists. Alpha values were at 1% cut-off 0.83 (TMA-A) and 0.66 (TMA-B), and at 50% cut-off 0.77 (TMA-A) and 0.78 (TMA-B). Interlaboratory variability of staining was higher (pConclusionsThe results provide insight in factors contributing to variability of immunohistochemical assessment of PD-L1, and contribute to more reliable predictive testing for PD-L1.

Background Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. Methods Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). Results In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. Conclusions A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

AimsMethods and resultsMost validation studies on digital pathology diagnostics have been performed in single institutes. Because rapid consultation on cases with extramural experts is one of the most important uses for digital pathology laboratory networks, the aim of this study was to validate a whole-slide image-based teleconsultation network between three independent laboratories.Each laboratory contributed 30 biopsies and/or excisions, totalling 90 specimens (776 slides) of varying difficulty and covering a wide variety of organs and subspecialties. All slides were scanned centrally at x40 scanning magnification and uploaded, and subsequently assessed digitally by 16 pathologists using the same image management system and viewer. Each laboratory was excluded from digital assessment of their own cases. Concordance rates between the two diagnostic modalities (light microscopic versus digital) were compared. Loading speed of the images, zooming latency and focus quality were scored. Leaving out eight minor discrepancies without any clinical significance, the concordance rate between remote digital and original microscopic diagnoses was 97.8%. The two cases with a major discordance (for which the light microscopic diagnoses were deemed to be the better ones) resulted from a different interpretation of diagnostic criteria in one case and an image quality issue in the other case. Average scores for loading speed of the images, zooming latency and focus quality were 2.37 (on a scale up to 3), 2.39 (scale up to 3) and 3.06 (scale up to 4), respectively.ConclusionsThis validation study demonstrates the suitability of a teleconsultation network for remote digital consultation using whole-slide images. Such networks may contribute to faster revision and consultation in pathology while maintaining diagnostic standards.

International audience; Biomarker analysis has become routine practice in the treatment of non-small cell lung cancer (NSCLC). To ensure high quality testing, participation to external quality assessment (EQA) schemes is essential. This article provides a longitudinal overview of the EQA performance for EGFR, ALK, and ROS1 analyses in NSCLC between 2012 and 2015. The four scheme years were organized by the European Society of Pathology according to the ISO 17043 standard. Participants were asked to analyze the provided tissue using their routine procedures. Analysis scores improved for individual laboratories upon participation to more EQA schemes, except for ROS1 immunohistochemistry (IHC). For EGFR analysis, scheme error rates were 18.8%, 14.1% and 7.5% in 2013, 2014 and 2015 respectively. For ALK testing, error rates decreased between 2012 and 2015 by 5.2%, 3.2% and 11.8% for the fluorescence in situ hybridization (FISH), FISH digital, and IHC subschemes, respectively. In contrast, for ROS1 error rates increased between 2014 and 2015 for FISH and IHC by 3.2% and 9.3%. Technical failures decreased over the years for all three markers. Results show that EQA contributes to an ameliorated performance for most predictive biomarkers in NSCLC. Room for improvement is still present, especially for ROS1 analysis.

Purpose: ALK rearrangement detection using FISH is the standard test to identify patients with non–small cell lung carcinoma (NSCLC) eligible for treatment with ALK inhibitors. Recently, ALK protein expression in resectable NSCLC showed predictive value. We evaluated tumor response rate and survival after crizotinib treatment of patients with advanced NSCLC with ALK activation using both dichotomous immunohistochemical (IHC) staining and FISH. Experimental Design: Patients with stage IV NSCLC treated with crizotinib were selected. Tumor response was assessed. ALK rearrangements were detected by FISH (Vysis ALK-break-apart FISH-Probe KIT) and IHC [Ventana ALK (D5F3) CDx assay]. Cohorts of patients with ALK-FISH–positive advanced NSCLC from four other hospitals were used for validation. Results: Twenty-nine consecutive patients with ALK-positive advanced NSCLC diagnosed by FISH and/or IHC on small biopsies or fine-needle aspirations (FNA) were treated with ALK inhibitors. All ALK-IHC–positive patients responded to crizotinib except three with primary resistance. No tumor response was observed in 13 ALK-FISH–positive but ALK-IHC–negative patients. This was confirmed in an external cohort of 16 patients. Receiver operator characteristic (ROC) curves for ALK-IHC and ALK-FISH compared with treatment outcome showed that dichotomous ALK-IHC outperforms ALK-FISH [tumor response area under the curve: (AUC), 0.86 vs. 0.64, P = 0.03; progression-free survival (PFS): AUC 0.86 vs. 0.36, P = 0.005; overall survival (OS): AUC, 0.78 vs. 0.41, P = 0.01, respectively]. Conclusions: Dichotomous ALK-IHC is superior to ALK-FISH on small biopsies and FNA to predict tumor response and survival to crizotinib for patients with advanced NSCLC. Our data strongly suggest adapting the guidelines and using dichotomous ALK-IHC as standard companion diagnostic test to select patients with NSCLC who benefit from ALK-targeting therapy. Clin Cancer Res; 23(15); 4251–8. ©2017 AACR.

In non-small-cell lung cancer higher PD-L1 expression of tumor cells is associated with a better response to immunotherapy1 . In routine clinical practice a substantial number of thoracic oncologists tend to use cytological techniques like fine needle aspirations (FNA) more often than biopsies. Cytopathological analysis of pleural fluid, trans-esophageal or bronchial FNA and bronchial washing is frequently performed allowing tumor typing as well as a concise evaluation for treatment targets. This article is protected by copyright. All rights reserved.

A 62-year-old man was referred to our university hospital for treatment of advanced adenocarcinoma of the lung after disease progression on two lines of EGFR TKI and one line of chemotherapy. Fluorescent in situ hybridization analysis upon progression showed an HER2 amplification. At our weekly Molecular Tumor Board (MTB), a decision was made to treat this patient with afatinib, which resulted in a partial response. However, progression was observed with a facial nerve paresis due to a metastasis in the skull. A biopsy of a location in the thorax revealed the presence of an EGFR-T790M mutation associated with acquired resistance, after which treatment with osimertinib was started. After 6 months, disease progression was observed, and a new biopsy was taken from the pelvic bone, which revealed the original amplification of HER2 together with the EGFR-L858R mutation, the EGFR-T790M mutation was not detected. The MTB decided to treat the patient with trastuzumab/paclitaxel. A partial response was observed in different bone lesions, while the skull metastasis with ingrowth in the brain remained stable for 6 months. Because of progression of the bone metastases after 6 months, a biopsy of a lesion in the thorax wall was taken. In this lesion, the EGFR-T790M mutation could be detected again. The MTB advised to start treatment with a combination of osimertinib and afatinib. This resulted in an impressive clinical improvement and a partial response of the bone metastases on the most recent 18-fluorodeoxyglucose positron emission tomography and computer tomography-scan. In conclusion, adjusting treatment to the mutational make-up of the tumor is a great challenge. For optimal treatment response multiple biopsies and re-biopsy upon progression are imperative. As more genes are investigated, treatment decision becomes increasingly difficult, therefore, expert opinions from an MTB is essential.

BACKGROUND: Predictive biomarkers allow clinicians to optimise cancer treatment decisions. Therefore, molecular biomarker test results need to be accurate and swiftly available. To ensure quality of oncology biomarker testing, external quality assessments (EQA) for somatic variant analyses were organised. This study hypothesised whether laboratory characteristics influence the performance of laboratories and whether these can be imposed before authorisation of biomarker testing. METHODS: Longitudinal EQA data from the European Society of Pathology were available over six (metastatic colorectal cancer) and four years (non-small cell lung cancer), including the percentage of analysis errors and technical failures, and information on laboratory characteristics (accreditation status, laboratory setting, number of samples analysed and detection method). Statistical models for repeated measurements were used to analyse the association between the EQA results and the laboratory characteristics. RESULTS: Laboratory accreditation was associated with fewer analysis errors in early stages of biomarker introduction into the laboratory. Analysing more samples, or university and research laboratories showed better performance. Changing the detection method did not have an effect. CONCLUSION: The indicators support the clinicians in choosing molecular pathology laboratories by improving quality assurance and guaranteeing patient safety. Accreditation of laboratories, centralisation of biomarker testing or a university and research setting should be stimulated. ispartof: BRITISH JOURNAL OF CANCER vol:119 issue:5 pages:605-614 ispartof: location:England status: Published online

Introduction: PD-L1 expression has been approved as predictive biomarker for immune checkpoint inhibitors (ICI) in non-small cell lung carcinoma (NSCLC). In case of lack of PD-L1 expression in tumor biopsies, 8-10% of patients will respond to ICI. However, in case of > 50% PD-L1 tumor expression half of patients show no response. Tumor immune escape phenotypes are characterized by loss of MHC-class-I and a lower grade of CD8 T-cell infiltration, despite upregulation of PD-L1 expression. At least one of the four phenotypes of MHC-class-I and PD-L1 co-expression is considered responsible for non-responsiveness. We investigated the frequency of PD-L1, MHC-class-I β2-microglobulin (β-2M) co-expression and presence of CD8 cells in advanced NSCLC. Methods: Analysis was performed on formalin fixed, paraffin embedded (FFPE) transbronchial or transthoracic tumor biopsies of 27 patients with advanced NSCLC. Immunohistochemistry was performed using Ventana Benchmark Ultra immunostainer. PD-L1 was detected using Ventana PD-L1 (SP263) Assay (CE IVD), CD8 by Ventana CONFIRM anti-CD8 (SP57) antibody and MHC-class I β2-M by Agilent-Dako polyclonal rabbit anti-human beta-2-Microglobulin. PDL1 expression was scored as percentage positive tumor cells according to the manufacturers guideline, β2-M was scored as positive or negative in tumor cells and CD8 cells as low (+), intermediate (++) or high (+++) numbers in tumor or peritumoral stroma. Results: In 27 patients IHC staining was performed. PDL1 expression was scored as 1%) patients had positive β-2M in 10 of 10 cases. PD-L1 negative tumors showed presence of β-2M in 10 of 17 cases. Seven out of 27 cases (26%) were β-2M negative; all of them were PD-L negative cases. Low CD8 score was seen in 7 patients and 2 of these patients have a PD-L1 expression >1% and positive β-2M. Conclusion: In this ongoing study one quarter of advanced NSCLC patients had no tumor MHC-class-1 β-2M expression. In our cohort β-2M negative phenotype was associated with lack of PD-L1 tumor expression. Citation Format: Lucie Hijmering-Kappelle, Jeroen Hiltermann, Birgitta Hiddinga, Anthonie van der Wekken, Nils 't Hart, Harry Groen, Wim Timens. PD-L1 expression, CD8 cells and MHC-class-1 Beta-2-microglobulin expression in advanced non-small lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4067.

Introduction:Patients with stage IV non-small-cell lung cancer harboring an activating epidermal growth factor receptor (EGFR) mutation are eligible for treatment with EGFR tyrosine kinase inhibitors. With pyrosequencing, low-frequency mutations may be detected more easily even in small diagnostic samples like endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspirations (EBUS-TBNA). The diagnostic performance of pyrosequencing in analyzing cytological specimens is compared with the routinely used high-resolution melting (HRM) and Sanger sequencing.Methods:Patients diagnosed with adenocarcinoma of the lung were selected from a fine needle aspiration and transbronchial needle aspiration specimen database. If formalin-fixed paraffin-embedded tumor blocks were available, mutation analysis was performed for EGFR and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes using both pyrosequencing and HRM. When HRM showed abnormalities, Sanger sequencing was used.Results:A total of 126 samples were available for mutation analysis. The analysis success rate for pyrosequencing and HRM were 97% and 93%, respectively. HRM failures were observed in fragmented DNA showing chains of 100 to 200bp. A significant correlation between length of DNA fragments (100-300bp versus 300-400bp) and mean sample age (797 versus 317 days) was found (p Conclusion:Pyrosequencing on cytological blocks, especially older tumor blocks, is feasible with a high diagnostic success rate. Failures in HRM were observed in DNA samples with short fragments related to longer storage times.

Hypothermic machine perfusion (HMP) provides better protection against ischemic damage of the kidney compared to cold-storage. The required perfusion pressures needed for optimal HMP of the liver are, however, unknown. Rat livers were preserved in University of Wisconsin organ preservation solution enriched with acridine orange (AO) to stain viable cells and propidium iodide (PI) to detect dead cells. Perfusion pressures of 12.5%, 25% or 50% of physiologic perfusion pressures were compared. Intravital fluorescence microscopy was used to assess liver perfusion by measuring the percentage of AO staining. After 1-h, the perfusion pressure of 12.5% revealed 72% +/- 3% perfusion of mainly the acinary zones one and two. The perfusion pressure of 25% and 50% showed complete perfusion. Furthermore, 12.5% showed 14.7 +/- 3.6, 25% showed 3.7 +/- 0.9, and 50% showed 11.2 +/- 1.4 PI positive cells. One hour was followed by another series of experiments comprising 24-h preservation. In comparison with 24-h cold-storage, HMP at 25% showed less PI positive cells and HMP at 50% showed more PI positive cells. In summary, perfusion at 25% showed complete perfusion, demonstrated by AO staining, with minimal cellular injury, shown with PI. This study indicates that fine-tuning of the perfusion pressure is crucial to balance (in)complete perfusion and endothelial injury.

Objective: To study graft function and ischemia/reperfusion injury of porcine kidneys after preservation with the new Groningen Machine Perfusion (GMP) system versus static cold storage (CS).Introduction: The increasing proportion of marginal and nonheart beating donors necessitates better preservation methods to maintain adequate graft viability. Hypothermic machine preservation (HMP) is a promising alternative to static CS. We have therefore developed and tested an HMP device, which is portable and actively oxygenates the perfusate via an oxygenator. The aim of the present study was to examine the efficacy of the GMP system in a transplantation experiment.Materials and Methods: In a porcine autotransplantation model, kidneys were retrieved and either cold stored in University of Wisconsin CS for 20 hours at 4 degrees C or subjected to HMP using University of Wisconsin machine perfusion at 4 degrees C with 2 different pressure settings: 30/20 mm Hg or 60/40 mm Hg.Results: HMP at 30/20 mm Hg was found to better preserve the viability of kidneys reflected by improved cortical microcirculation, less damage to the proximal tubule, less damage mediated by reactive oxygen species, less proinflammatory cytokine expression, and better functional recovery after transplantation. However, high perfusion pressures (60/40 mm Hg) resulted in higher expression of von Willebrand factor and monocyte chemotactic peptide-1 in post-preservation biopsies and subsequent graft thrombosis in 2 kidneys.Conclusions: It is concluded that the GMP system improves kidney graft viability and perfusion pressures are critically important for outcome.

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea-synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia-reperfusion injury. Furthermore, expression of uncoupling protein-2 (UCP-2) mRNA was assessed since UCP-2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW-MP) with 0%, 21%, or 95% oxygen at 0-4 degrees C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW-MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea-synthesis rate as well as lower ATP levels. mRNA upregulation of UCP-2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW-MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP-2. We conclude that saturation of UW-MP with 21% oxygen allows optimal preservation results.

Organ procurement is the first step toward effective liver preservation and comprises a thorough washout of blood components from the microvasculature. To study the efficacy of optimal blood washout of the liver, three groups were compared including low-pressure perfusion with UW-CSS (12 mmHg, group A), which is the routine method in clinical practice, high-pressure perfusion with UW-CSS (100 mmHg, group B) and low-pressure perfusion with modified UW solution (12 mmHg, group C). After procurement all livers were preserved in original UW-CSS for 0, 24 or 48 h, followed by reperfusion in oxygenated Williams Medium E for 24 h at 37 degrees C. Histology results of livers procured in group A, showed good hepatocyte viability but also remaining erythrocytes. However, injury parameters were high and ATP concentrations were low. No functional differences were found. Group B, high pressure, and group C, modified UW-CSS, both showed better results. High-pressure washout is preferable since the warm ischemia time during procurement is short. We propose to use high-pressure UW-CSS perfusion for the initial blood washout of the donor liver instead of the usually used low-pressure washout.

To overcome the present shortage of liver donors by expansion of the existing donor pool and possibly lengthening of the storage time, hypothermic machine perfusion of the liver as a dynamic preservation method is revisited. The three most important aspects are defined to be the type of preservation solution, the characteristics of perfusion dynamics, and the oxygen supply. Reviewing hypothermic liver machine perfusion experiments, the University of Wisconsin machine preservation solution is the solution most used. It is also found that nothing conclusive can be said about the optimal perfusion characteristics, since either perfusion pressure or perfusion flow is reported. The best estimation is perfusion of the liver in a physiological manner, i.e. pulsatile arterial perfusion and continuous portal venous perfusion. The applied pressures could be chosen to be somewhat lower than physiological pressures to prevent possible endothelial cell damage. Oxygen supply is necessary to achieve optimal preservation of the liver. The minimal amount of partial oxygen pressure required is inversely related to the normalized flow. Incorporating these features in a system based on existing standard surgical and organ sharing procedures and which is able to work stand-alone for 24 It, weighing less than 23 kg, could successfully implement this technique into every day clinical practise.

Introduction: Breakthroughs in cancer research have resulted in mutation-specific targeted therapies (precision medicine). Most of these new drugs are only effective in patients with an actionable molecular profile. Thus, predictive molecular testing enables oncologists to select individual patients for the most appropriate (targeted) therapy and to reduce the burden of overtreatment. The number of clinically relevant predictive markers that are routinely analyzed is growing rapidly, resulting in the identification of rare mutations, mutations with unknown relevance and coexistence of two or more mutations in the same sample. Incorporating these into the optimal treatment for the individual patient can be complex. Methods: A total of 2461 sequential tumor biopsies were analyzed at our institute using targeted next generation sequencing (Ion Torrent platform). 230 of these patients were discussed at a weekly Molecular Tumor Board (MTB) meeting. Cases involved 170 lung and 21 colorectal carcinomas, 24 melanomas, 1 GIST and a range of other malignancies with uncommon and rare mutations. The board is composed of pulmonologists, medical oncologists, pathologists and clinical scientists in molecular pathology. The goal of the MTB is to discuss the biological and clinical relevance of rare mutations or uncommon profiles and to suggest treatment options based on registered, off-label or trial-based drugs presently available in the Netherlands. Results: In this abstract we report on four patients with an ERBB2 exon 20 mutation and 1 patient with ERBB2 amplification received anti-HER2 treatment after an MTB consensus decision. Two patients with an insertion in exon 20 of ERBB2: (c.2313_2324dup; p.(Y772_A775dup)) received first line therapy with afatinib and showed a partial response and stable disease respectively. One patient with a c.2524G>A; p.(V842I) mutation received afatinib and showed stable disease for 3 months. A patient with another ERBB2 exon 20 insertion (c.2326_2327insTAT:p.(G776delinsVC)) received afatinib but had progressive disease within two months. One patient with an ERBB2 amplification by FISH and high (3+) HER2(ERBB2) expression, showed a partial response to trastuzumab. All patients had stage IV and would without genomic knowledge been treated with chemotherapy. Conclusion: Lung cancer patients with sporadic ERBB2 mutations might benefit from targeted ERBB2 therapy. For an optimal treatment decision, patients with rare mutations in general, may benefit from discussion in a multidisciplinary molecular tumor board. In the future, both the considerations for targeted therapy as well as treatment response and toxicity should be registered in an open-access database and shared with other national and international Molecular Tumor Board initiatives to allow comparison with traditional treatments. Citation Format: Arja ter Elst, Nils A. 't Hart, Anthonie J. van der Wekken, Wim Timens, Lucie B. Hijmering-Kappelle, Geke A. Hospers, Hilde Jalving, Elise M. van der Logt, Leon C. van Kempen, Sjoukje F. Oosting, Matthew R. Groves, T Jeroen Hiltermann, Anke van den Berg, Harry J. Groen, Ed Schuuring. Treatment decision-making of rare ERBB2 (HER2) mutations in lung cancer; a role for multidisciplinary molecular tumor boards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 754. doi:10.1158/1538-7445.AM2017-754

Purpose: To evaluate treatment response following the Multidisciplinary Molecular Tumor Board (MTB) decisions which focused on EGFR exon 20 mutations in lung adenocarcinoma. Methods: Molecular studies were routinely performed using the Ion Torrent sequencing platform for histologically or cytologically diagnosed lung adenocarcinoma. Since October 2014 patients with rare (prevalence Summary of data: Over a period of 2 years 1389 samples were tested. A total of 170 (12.2%) rare mutations were detected in lung cancer. Among these rare mutations we observed 16 EGFR exon 20 insertions and other mutations like e.g: p.T790M, p.D761N, p.D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, p.N771_H773dup, p.T790S. Using an in silico modeling of protein responses with TKI were predicted to be likely in EGFR T790M mutations and p.S768_V769delinsIL, but not in p. D770delinsGY, p.S768_D770dup, p.V769_D770insSFL, and p.N771_H773dup mutations. Eight patients with an exon 20 mutation were treated with an EGFR TKI. One patient (p.D761N) had a partial response on erlotinib 300mg daily. On afatinib monotherapy 2 out of 3 pts had stable disease (PFS 3-11 months). On afatinib/cetuximab treatment 1 out of 2 pts (p.D770delinsGY) had a partial response and the other patient had stable disease (p.S768_D770dup) (PFS 11 and 4 months, resp.). Two evaluable patients were treated with osimertinib: the first patient initially progressed on afatinib (p.V769_D770insSFL). The protein model predicted no response, since we predicted hindrance of osimertinib. The model predicted no response on osimertinib in the second patient as well, and during treatment a stable disease for 4 months was observed (p.N771_H773dup). This molecular information from the weekly MTB meeting was delivered within 2 weeks to the treating physician. Feedback on treatment outcome helped to further improve treatment predictions. Conclusion: The Molecular Tumor Board is an effective multidisciplinary team to discuss rare mutations. Our pilot data show that the evaluation of the use and effectiveness of a theoretical model concerning protein structures is not possible yet, however, did provide a clear insight in protein structures in a mutated EGFR receptor supporting decision making of treatment options. Citation Format: Anthonie J. van der Wekken, Matthew R. Groves, Arja ter Elst, Nils A. 't Hart, Lucie B. Hijmering-Kappelle, Thijo J. Hiltermann, Anke van den Berg, Wim Timens, Ed Schuuring, Harry J. Groen. Molecular Tumor Board treatment predictions on rare EGFR exon 20 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2017-2718

Background. Brain death donors are frequently used for transplantation. Previous studies showed that brain death (BD) negatively affects the immunological and inflammatory status of both liver and kidney. Because the intestine is increasingly used as a donor organ and no information on effects of BD on small intestine is available we performed this study.Methods. We studied the inflammatory and apoptotic changes in donor intestine after BD induction. Brain death was induced in rats by inflation of a balloon catheter. Three groups (n=6) were compared: 1-hr BD, 4-hr BD, and sham-operated controls.Results. An increased polymorphonuclear cell influx in ileum, as a measure of inflammation, was observed in 1- and 4-hr BD group compared with controls. Jejunum showed a significant increase at the 4-hr BD group compared with the control group. Intercellular adhesion molecule-1, vascular cell adhesion molecule- 1, E-selectin, and interleukin-6 were upregulated after 1- and 4-hr BD. Caspase-3 positive cells were found in jejunum and ileum after 4-hr BD on the top of the villi. Serum interleukin-6 was severely elevated in the 1- and 4-hr brain dead rats.Conclusion. These data show the early occurrence of intestinal inflammation and apoptosis after BD induction. These events may ultimately have a negative influence on the outcome of intestinal transplantation.

Organs used for transplantation are usually derived from heart-beating brain dead donors. However, brain death is known to have negative effects on donor organ quality, previously studied using a difficult to control sudden onset experimental model. We have now developed a reproducible gradual onset brain death model in rats without requiring inotropic support. Fisher inbred rats weighing 260–300 g were used. Brain death was induced by a gradual inflation of a subdurally placed balloon catheter. During induction and the period following brain death, the animals were mechanically ventilated and blood pressure was continuously monitored. The blood pressure registration showed a characteristic pattern during brain death induction, in which a decrease in blood pressure, a hypotensive period in which the Cushing response occurred, and a sharp peak were consistent findings. After brain death was induced, blood pressure was maintained at normotensive levels up to 4 h. After the experiments, neuropathological evaluation of the brain located haemorrhagic cerebral parenchyma, and immunocytochemistry of liver tissue revealed a significant influx of polymorph nuclear cells, as was previously observed as well. This improved model allows the study of brain death on donor organ quality without the use of inotropic support.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea-synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia-reperfusion injury. Furthermore, expression of uncoupling protein-2 (UCP-2) mRNA was assessed since UCP-2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW-MP) with 0%, 21%, or 95% oxygen at 0-4 degrees C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW-MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea-synthesis rate as well as lower ATP levels. mRNA upregulation of UCP-2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW-MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP-2. We conclude that saturation of UW-MP with 21% oxygen allows optimal preservation results.

Ischemic injury to the renal allograft prior to implantation is considered as the major cause of primary non and never-function (PNF) and delayed graft function (DGF). Evidence has been put forward that brain dead and non-heart-beating (NHB) donor organs are of marginal quality compared to living donors. The purpose of this study was to evaluate renal function and injury of brain dead and NHB donor kidneys using the isolated perfused rat kidney.Fisher F344 rats were either maintained brain death for 4 hr or subjected to cardiac arrest for 45 min (NHB). Living rats served as controls. To omit additional effects of cold ischemia, kidneys were immediately reperfused. Renal function and injury were assessed by monitoring urine production, glomerular filtration rate (GFR), Na+ and K+ reabsorption, glucose metabolism and reabsorption, as well as release of brush border, lysosomal, and intracellular enzymes.Renal dysfunction and injury were most pronounced in NHB donor kidneys reflected by a highly reduced urine production, anaerobic glucose metabolism resulting in lactate formation, and significant higher luminal release of intracellular and lysosomal enzymes. Brain dead kidneys showed an increased urine production and were functionally abnormal in K+ reabsorption showing a net excretion of K+, probably as a result of ATP depletion. Loss of brush border occurred during brain death and cardiac arrest.Both, brain death and cardiac arrest have deleterious effects on renal function and renal injury. The ischemically injured NHB donor kidney was functionally inferior compared to the brain dead donor kidney and living donor kidneys. However, both brain dead and NHB kidneys showed considerable renal damage compared to kidneys from living donors.

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 mu mol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 mu mol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.

In conventional cold-storage organ preservation, the donor organ is flushed with University of Wisconsin (UW) solution at 0-4 degrees C. The initial flush is used to wash out blood from the microcirculation to allow optimal preservation with the UW solution. The component hydroxyethyl starch (HES) of UW is known to cause relatively high viscosity and a possible interaction with blood, i.e. increased red blood cell (RBC) aggregation. The aim of this study was to investigate the influence of the HES component on the viscosity of UW and the aggregation behaviour of blood during washout. Viscosity aspects were measured with a cone-plate rheometer. HES-induced RBC aggregation was studied by means of an optical aggregation measuring device. The experiments were carried out with rat whole blood and mixtures of rat whole blood with UW-solution and UW without HES (UWmod), at 4 degrees C. The viscosity of blood at 4 degrees C is two-times higher than at 37 degrees C; the UW/blood mixture at 4 degrees C is 1.3-times more viscous than blood at 37 degrees C; the 4 degrees C UWmod/blood mixture equals the viscosity of blood at 37 degrees C. The UW/blood mixture shows a ninefold increased aggregation compared with whole blood. These aggregates are larger than the diameter of the sinusoids in the rat liver. A mixture of whole blood and UWmod shows a lower aggregation than blood. Apart from an increased viscosity, HES in UW causes increased RBC aggregation. The aggregates are larger than the diameter of the sinusoids. Initial washout could be optimised by pre-flushing to improve the viability of the liver and to decrease delayed graft function.

Background. The standard preservation solution used during organ procurement and preservation of most organs is the University of Wisconsin (UW) solution. Despite its superiority over other cold storage solutions, the inclusion of hydroxyethyl starch (HES) as one of the components of the UW solution has been both advocated and denied. This study determined whether HES had any effect on red blood cell (RBC) aggregability and correlated aggregation parameters with HES molecular weight.Methods. Human RBC aggregability and deformability were investigated in vitro, at 4degreesC, with a laser-assisted optical rotation cell analyzer. The study of RBC aggregation in a binary HES-HES system gave an indication about the nature of HES-RBCs interactions. Bright field microscopy and atomic force microscopy were used to morphologically characterize the aggregates size and form.Results. High molecular weight HES and UW solution had a potent hyperaggregating effect; low molecular weight HES had a hypoaggregating effect on RBC. RBC aggregates were of large size and their resistance to dissociation by flow-induced shear stress was high.Conclusion. The authors' in vitro experiments conclusively showed that the physiologic function of RBCs to form aggregates is significantly affected in the presence of HES. The use of high molecular weight HES in UW solution accounts for extended and accelerated aggregation of erythrocytes that may result in stasis of blood and incomplete washout of donor organs before transplantation.