Birgit Weynand

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v‐ras] oncogene homolog (NRAS), B‐Raf proto‐oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit α (PIK3CA). Next‐generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single‐institution studies rather than multicenter experiences.

Phenotype molding of stromal cells in the lung tumor microenvironment

Cancer cells are embedded in the tumor microenvironment (TME), a complex ecosystem of stromal cells. Here, we present a 52,698-cell catalog of the TME transcriptome in human lung tumors at single-cell resolution, validated in independent samples where 40,250 additional cells were sequenced. By comparing with matching non-malignant lung samples, we reveal a highly complex TME that profoundly molds stromal cells. We identify 52 stromal cell subtypes, including novel subpopulations in cell types hitherto considered to be homogeneous, as well as transcription factors underlying their heterogeneity. For instance, we discover fibroblasts expressing different collagen sets, endothelial cells downregulating immune cell homing and genes coregulated with established immune checkpoint transcripts and correlating with T-cell activity. By assessing marker genes for these cell subtypes in bulk RNA-sequencing data from 1,572 patients, we illustrate how these correlate with survival, while immunohistochemistry for selected markers validates them as separate cellular entities in an independent series of lung tumors. Hence, in providing a comprehensive catalog of stromal cells types and by characterizing their phenotype and co-optive behavior, this resource provides deeper insights into lung cancer biology that will be helpful in advancing lung cancer diagnosis and therapy.A comprehensive single-cell analysis of the lung cancer microenvironment reveals marked heterogeneity of transcriptional networks that defines novel clinically relevant stromal cell populations.

Effectiveness of crizotinib in a patient with ALK IHC-positive/FISH-negative metastatic lung adenocarcinoma.

We report a case of crizotinib effectiveness in a heavily pretreated patient with a metastatic NSCLC initially considered IHC-positive and FISH-negative for ALK rearrangement. After repeated analyses of tumor samples, borderline ALK FISH-positivity (18.5% positive cells) was demonstrated.

Diagnostic Ability of a Dynamic Multidisciplinary Discussion in Interstitial Lung Diseases: A Retrospective Observational Study of 938 Cases

Background: The advice of a dynamic multidisciplinary discussion (MDD) is believed to be important in the diagnosis of interstitial lung diseases (ILDs). However, to what extent MDD diagnoses differ from the preliminary diagnoses before formal workup and MDD (preMDD diagnoses) is still insufficiently studied. Methods: We compared preMDD and MDD diagnoses in patients discussed at the Leuven University Hospitals MDDs between January 2005 and December 2015. Results: Of 938 consecutive patients discussed in an MDD, 755 (80.5%) received a specific diagnosis. From the 183 patients with unclassifiable ILD, 150 patients (16.0%) received suggestions concerning further investigations to establish a definite diagnosis. In 191 patients (41.9% of patients with a preMDD diagnosis), the MDD changed the diagnosis. In 384 patients (79.5% of patients without preMDD diagnosis), MDD provided a diagnosis when the referring physician did not. MDD diagnosis showed a trend toward better prognostic discrimination between idiopathic pulmonary fibrosis and other ILDs compared with preMDD diagnosis (Harrell C‐index, 0.666 vs 0.631; P = .08), which was particularly clear in patients with discordant MDD and preMDD diagnoses (hazard ratio, 2.68 vs 0.84; P = .012 vs .768). Conclusions: The MDD provided a definite diagnosis in 80.5% of presented cases, suggesting further investigations in almost all others. Given the high number of patients without preMDD diagnosis, the rate of change in preMDD diagnoses (41.9% of patients with a preMDD diagnosis) probably is an underestimation. The better prognostic discrimination among ILDs by using MDD indicates the added value of MDD in ILD.

Nintedanib reduces radiation -induced microscopic lung fibrosis but this cannot be monitored by CT imaging: A preclinical study with a high precision image-guided irradiator

BACKGROUNDnNintedanib has anti-fibrotic and anti-inflammatory activity and is approved for the treatment of idiopathic pulmonary fibrosis. The aim of this study was to noninvasively assess the efficacy of nintedanib in a mouse model of partial lung irradiation to prevent radiation-induced lung damage (RILD).nnnMETHODSn266 C57BL/6 adult male mice were irradiated with a single radiation dose (0, 4, 8, 12, 16 or 20Gy) using parallel-opposed fields targeting the upper right lung using a precision image-guided small animal irradiator sparing heart and spine based on micro-CT images. One week post irradiation, mice were randomized across nintedanib daily oral gavage treatment (0, 30 or 60mg/kg). CT density analysis of the lungs was performed on monthly acquired micro-CT images. After 39weeks, lungs were processed to evaluate the fibrotic phenotype.nnnRESULTSnAlthough the CT density increase correlated with the radiation dose, nintedanib did not influence this relationship. Immunohistochemical analysis confirmed the ability of nintedanib to reduce the microscopic fibrotic phenotype, in particular interstitial edema, interstitial and perivascular fibrosis and inflammation, and vasculitis.nnnCONCLUSIONSnNintedanib reduces radiation-induced lung fibrosis after partial lung irradiation without adverse effects, however, noninvasive CT imaging measuring electron density cannot be applied for monitoring its effects.

The challenge of molecular testing for clinical trials in advanced non-small cell lung cancer patients: Analysis of a prospective database

OBJECTIVESnMolecular testing has become important in the biomarker program of clinical trials for advanced non-small lung cancer (NSCLC). These tissue samples often have to be analyzed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process and how often testing resulted in inclusion in a clinical trial.nnnMETHODSnWe reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 and October 31, 2014.nnnRESULTSn250 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis and 20 patients had failure of central biomarker analysis. Results were obtained for 207 (83%) patients. In 91 of 227 (40%) samples sent, a biomarker of interest was documented. This led to 34 (15%) clinical trial inclusions. The mean waiting time between informed consent and request for tissue sections from the pathology lab and receipt of biomarker result from central lab was 24.4 (SD 13.7) calendar days.nnnCONCLUSIONnWhile molecular biomarker testing is crucial in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients.

Roles of inflammation and apoptosis in experimental brain death-induced right ventricular failure.

BACKGROUNDnRight ventricular (RV) dysfunction remains the leading cause of early death after cardiac transplantation. Methylprednisolone is used to improve graft quality; however, evidence for that remains empirical. We sought to determine whether methylprednisolone, acting on inflammation and apoptosis, might prevent brain death-induced RV dysfunction.nnnMETHODSnAfter randomization to placebo (n = 11) or to methylprednisolone (n = 8; 15 mg/kg), 19 pigs were assigned to a brain-death procedure. The animals underwent hemodynamic evaluation at 1 and 5 hours after Cushing reflex (i.e., hypertension and bradycardia). The animals euthanized, and myocardial tissue was sampled. This was repeated in a control group (n = 8).nnnRESULTSnAt 5 hours after the Cushing reflex, brain death resulted in increased pulmonary artery pressure (27 ± 2 vs 18 ± 1 mm Hg) and in a 30% decreased ratio of end-systolic to pulmonary arterial elastances (Ees/Ea). Cardiac output and right atrial pressure did not change. This was prevented by methylprednisolone. Brain death-induced RV dysfunction was associated with increased RV expression of heme oxygenase-1, interleukin (IL)-6, IL-10, IL-1β, tumor necrosis factor (TNF)-α, IL-1 receptor-like (ST)-2, signal transducer and activator of transcription-3, intercellular adhesion molecules-1 and -2, vascular cell adhesion molecule-1, and neutrophil infiltration, whereas IL-33 expression decreased. RV apoptosis was confirmed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling staining. Methylprednisolone pre-treatment prevented RV-arterial uncoupling and decreased RV expression of TNF-α, IL-1 receptor-like-2, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neutrophil infiltration. RV Ees/Ea was inversely correlated to RV TNF-α and IL-6 expression.nnnCONCLUSIONSnBrain death-induced RV dysfunction is associated with RV activation of inflammation and apoptosis and is partly limited by methylprednisolone.

An Appraisal of Proliferation and Apoptotic Markers in Papillary Thyroid Carcinoma: An Automated Analysis.

Introduction Proliferation and apoptosis are opposing processes by which the cell numbers are kept in a delicate balance, essential for tissue homeostasis, whereas uncontrolled growth of cells is a hallmark of cancer. Papillary thyroid cancer (PTC) is the commonest type of thyroid cancer, with some PTC following an indolent course, whereas the other ones are more aggressive. Aim To evaluate respective contribution of proliferation and apoptosis in the tumorigenesis of PTC by automated analysis. Materials and Methods We investigated the immunolabeling of phosphorylated histone H3 (pHH3), cyclin D1, active caspase-3, and bcl-2 in thirteen cases each of metastatic PTC, follicular variant of PTC (FVPTC), papillary microcarcinoma (PMC) and well differentiated tumor of uncertain malignant potential (WDT-UMP). FVPTC cases comprised seven encapsulated and six unencapsulated cases. Results Proliferation, as assessed by pHH3 and cyclin D1 immunolabeling, was increased in all PTC variants, including the putative precursor lesion WDT-UMP, compared to normal thyroid tissue. pHH3 was immunolabeled in more cells of metastatic PTC than of PMC and of encapsulated FVPTC. Surprisingly, metastatic PTC and unencapsulated FVPTC also demonstrated more cleaved caspase-3 immunolabeled cells than the other types. In contrast, increased expression of bcl-2 protein was seen in normal thyroid areas, encapsulated FVPTC and PMC as compared to metastatic PTC. Metastatic PTC shows higher proliferation than other types of PTC but unexpectedly also higher apoptotic levels. Similar results were also seen with unencapsulated FVPTC, thus suggesting that unencapsulated FVPTC has a potential for adverse outcome. Bcl-2 was immunolabeled in a low percentage of cells in WDT-UMP. Conclusions The expression of the proliferative protein pHH3 together with the apoptotic marker cleaved caspase-3 may indicate an aggressive behaviour of PTC and loss of apoptosis inhibition by bcl-2 protein can further amplify the role of these proteins in tumor progression. Both cyclin D1 and bcl-2 could prove to be interesting markers of PTC precursor lesions. Automated/digital image quantification approach helps in refining the diagnostic accuracy.

The use of p16/Ki-67 dual staining technology on cervical cytology of patients undergoing a LLETZ procedure

OBJECTIVESnThe main objective of this prospective observational study was to investigate the diagnostic performance of the p16/Ki-67 dual stain technique (DST) for detecting CIN 2+ in a LLETZ referral setting. Test performances were compared with HR-HPV testing and Pap cytology.nnnMETHODSnAll patients referred for a LLETZ procedure were candidates for participation in this trial. A total of 110 patients were enrolled between October 2016 and March 2017. From each participant, a cervical cytology sample was obtained before the onset of the LLETZ procedure. On each sample, the dual stain technique (Roche CINtec PLUS ® test), Pap cytology and an HPV DNA assay (identifying 17 different HPV types) were performed.nnnRESULTSnThe overall disease prevalence of CIN 2+ was 56%. The mean age was 41 years, with 38% of patients being younger than 35 years. The overall sensitivity and specificity of the dual stain technique for detecting CIN 2+ was 94% (95% CI: 84.30-98.21%) and 58% (95% CI: 43.21-72.93%) respectively with a PPV of 74% (95% CI: 67.34-80.31%) and a NPV of 88% (95% CI: 72.48-94.90%). HR-HPV testing results in a similar sensitivity of 92% (95% CI: 82.17-97.33%) but considerable lower specificity of 21% (95% CI: 11.17-33.35%) compared to the dual stain technique. At an ASCUS or worse threshold, Pap cytology had the lowest sensitivity of 89% (95% CI 78.11-95.34%) compared to dual staining and HR-HPV testing. Specificity was better (48% with 95% CI of 33.29-62.81%) than that of HR-HPV testing but not as good as the DST.nnnCONCLUSIONnp16/Ki-67 dual staining provides high sensitivity and improved specificity compared to HR-HPV testing and Pap cytology for detecting CIN 2+, making it an interesting tool for identifying relevant disease in patients referred for a LLETZ procedure.

Radiological analysis of unused donor lungs: A tool to improve donor acceptance for transplantation?

Despite donor organ shortage, a large proportion of possible donor lungs are declined for transplantation. Criteria for accepting/declining lungs remain controversial because of the lack of adequate tools to aid in decision‐making. We collected, air‐inflated, and froze a large series of declined/unused donor lungs and subjected these lung specimens to CT examination. Affected target regions were scanned by using micro‐CT. Lungs from 28 donors were collected. Two lungs were unused, six were declined for non–allograft‐related reasons (collectively denominated nonallograft declines, n = 8), and 20 were declined because of allograft‐related reasons. CT scanning demonstrated normal lung parenchyma in only four of eight nonallograft declines, while relatively normal parenchyma was found in 12 of 20 allograft‐related declines. CT and micro‐CT examinations confirmed the reason for decline in most lungs and revealed unexpected (unknown from clinical files or physical inspection) CT abnormalities in other lungs. CT‐based measurements showed a higher mass and density in the lungs with CT alterations compared with lungs without CT abnormalities. CT could aid in the decision‐making to accept or decline donor lungs which could lead to an increase in the quantity and quality of lung allografts.