Monika G. Looijen-Salamon

A Brief Retrospective Report on the Feasibility of Epidermal Growth Factor Receptor and KRAS Mutation Analysis in Transesophageal Ultrasound- and Endobronchial Ultrasound-Guided Fine Needle Cytological Aspirates

Introduction: Molecular testing for epidermal growth factor receptor (EGFR) and KRAS mutations is of increasing clinical importance in daily practice. In this study, we aimed to investigate the yield and applicability of molecular testing for KRAS and EGFR mutations in cytologic specimens obtained by EUS or endobronchial ultrasound (EBUS)-guided fine needle aspiration (FNA). Methods: We selected all patients with an EUS- or EBUS-guided FNA positive for lung adenocarcinoma from the database of our tertiary care center for endosonography. Direct smears were Giemsa and Papanicolaou stained. The remaining material was processed in cell blocks. Both cell blocks and smears were considered suitable for molecular analysis when >40% of the aspirated cells were tumor cells. All eligible samples were investigated for KRAS and EGFR mutations by polymerase chain reaction followed by direct sequencing. Results: Four hundred sixty-two patients underwent EUS or EBUS-FNA using 22-gauge needles. In 35 patients, FNA showed lung adenocarcinoma. In eight patients, molecular analysis could not be performed because of insufficient material after routine and immunocytochemistry (n = 3), a low percentage (<40%) of tumor cells (n = 3), or an insufficient DNA quality (n = 2). The average percentage of tumor cells was 73% ± 23%. Molecular analysis could reliably be performed in 27 patients (77%). Mutation analysis showed KRAS and EGFR mutations in tumor samples from 10 (37%) and two (7%) patients, respectively. In one patient, two EGFR mutations (p.Thr790Met and p.Leu858Arg) were detected. Conclusions: Molecular analysis for KRAS and EGFR mutations can be performed routinely in cytologic specimens from EUS- and EBUS-guided FNA.

Differences in metabolism between adeno- and squamous cell non-small cell lung carcinomas: spatial distribution and prognostic value of GLUT1 and MCT4.

BACKGROUND Hypoxia leads to changes in tumor cell metabolism such as increased glycolysis. In this study, we examined the spatial distribution of the glycolysis and hypoxia related markers glucose transporter 1 (GLUT1) and monocarboxylate transporter 4 (MCT4) expression in relation to the vasculature in stage I, II and resectable stage IIIA NSCLC. Furthermore, associations of these markers with survival were investigated. METHODS GLUT1 and MCT4 expression were determined in 90 NSCLC fresh frozen biopsies using immunohistochemical techniques and a computerized image analysis system. Markers were analyzed for adenocarcinomas (n=41) and squamous cell carcinomas (n=34) separately. Eighty-four patients were retrospectively evaluated for relapse and survival. RESULTS Squamous cell carcinomas demonstrated higher GLUT1 expression, relative to adenocarcinomas. Also, in squamous cell carcinomas, GLUT1 and MCT4 expression increased with increasing distance from the vasculature, whereas in adenocarcinomas upregulation of MCT4 was already found at closer distance from vessels. In adenocarcinomas, high GLUT1 expression correlated with a poor differentiation grade and positive lymph nodes at diagnosis. High GLUT1 plus high MCT4 expression was associated with a poor disease-specific survival in only adenocarcinomas (p=0.032). CONCLUSION Analysis of GLUT1 and MCT4 expression on the histological level suggested a different metabolism for adenocarcinomas and squamous cell carcinomas. Likely, adenocarcinomas rely mainly on aerobic glycolysis for ATP production, whereas the behavior of squamous cell carcinomas is more physiologically, i.e. mitochondrial oxidation with anaerobic glycolysis under hypoxic conditions. High GLUT1 plus high MCT4 expression indicated an aggressive tumor behavior in adenocarcinomas. This subgroup of tumors may benefit from new treatment approaches, such as MCT4 inhibitors. Since this study has an exploratory character, our results warrant further investigation and need independent validation.

Glucose Metabolism in NSCLC Is Histology-Specific and Diverges the Prognostic Potential of 18FDG-PET for Adenocarcinoma and Squamous Cell Carcinoma

Introduction: Biological features of non–small-cell lung carcinomas (NSCLCs) are important determinants for prognosis. In this study, differences in glucose metabolism between adeno- and squamous cell NSCLCs were quantified using the hypoxia and glycolysis-related markers glucose transporter 1 (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 (MCT1) and 4 (MCT4) vasculature, and 18-fluoro-2-deoxyglucose (18FDG)-uptake. Relevance of these markers for disease-free survival (DFS) was analyzed. Methods: Patients with curatively resected stage I to II and resectable stage IIIA, cN0-1 adeno- or squamous cell NSCLC, of whom fresh-frozen lung resection biopsies and pretreatment 18FDG-positron emission tomography (PET) scans were available, were included in this study (n = 108). 18FDG-uptake was quantified by calculating total lesion glycolysis (TLG). Metabolic marker expression was measured by immunofluorescent staining (protein) and quantitative polymerase chain reaction (messenger ribonucleic acid [mRNA]). Patients were retrospectively evaluated for DFS. Results: mRNA and protein expression of metabolic markers, with the exception of MCT4, and TLG were higher in squamous cell carcinomas than in adenocarcinomas, whereas adenocarcinomas were better vascularized. Adenocarcinomas had a worse DFS compared with squamous cell carcinomas (p = 0.016) based on the potential to metastasize. High TLG was associated with a worse DFS only in adenocarcinomas. Conclusion: Our findings suggest that the adenocarcinomas exhibit glycolysis under normoxic conditions, whereas squamous cell carcinomas are exposed to diffusion-limited hypoxia resulting in a very high anaerobic glycolytic rate. Although squamous cell carcinomas have a higher 18FDG-uptake, in general regarded as a poor prognostic factor, adenocarcinomas have a higher metastatic potential and a worse DFS. These findings show that 18FDG-PET should be interpreted in relation to histology. This may improve the prognostic potential of 18FDG-PET and may aid in exploiting 18FDG-PET in treatment strategies allied to histology.

Timeliness of lung cancer diagnosis and treatment in a rapid outpatient diagnostic program with combined 18FDG-PET and contrast enhanced CT scanning.

INTRODUCTION Delays in the diagnosis of lung cancer are under debate and may affect outcome. The objectives of this study were to compare various delays in a rapid outpatient diagnostic program (RODP) for suspected lung cancer patients with those described in literature and with guideline recommendations, to investigate the effects of referral route and symptoms on delays, and to establish whether delays were related to disease stage and outcome. METHODS A retrospective chart study was conducted of all patients with suspected lung cancer, referred to the RODP of our tertiary care university clinic between 1999 and 2009. Patient characteristics, tumor stage and different delays were analyzed. RESULTS Medical charts of 565 patients were retrieved. 290 patients (51.3%) were diagnosed with lung cancer, 48 (8.5%) with another type of malignancy, and in 111 patients (19.6%) the radiological anomaly was diagnosed as non-malignant. In 112 (19.8%) no immediate definite diagnosis was obtained, however in 82 of these cases (73.2%) the proposed follow-up strategy confirmed a benign outcome. The median first line delay was 54 days, IQR (interquartile range) 20-104 days, median patient delay 19 days (IQR 4-52 days), median referral delay was 7 days (IQR 5-9 days), median diagnostic delay 2 days (IQR 1-19 days). In 87% a diagnosis was obtained within 3 weeks after visiting a chest physician and 52.5% started curative therapy within 2 weeks after diagnosis. Patients presenting with hemoptysis had shorter first line delays. The RODP care was generally far more timely compared to literature and published guidelines, except for both referral and palliative therapeutic delay. No specific delay was significantly related to disease stage or survival. CONCLUSIONS An RODP results in a timely diagnosis well within guideline recommendations. Patient and first line delay account for most of total patient delay. Within the limitations of this retrospective study, we found no association with disease stage or survival.

The role of 18 F-FDG PET in the differentiation between lung metastases and synchronous second primary lung tumours

PurposeIn lung cancer patients with multiple lesions, the differentiation between metastases and second primary tumours has significant therapeutic and prognostic implications. The aim of this retrospective study was to investigate the potential of 18F-FDG PET to discriminate metastatic disease from second primary lung tumours.MethodsOf 1,396 patients evaluated by the thoracic oncology group between January 2004 and April 2009 at the Radboud University Nijmegen Medical Centre, patients with a synchronous second primary lung cancer were selected. Patients with metastatic disease involving the lungs served as the control group. Maximum standardized uptake values (SUVs) measured with 18F-FDG PET were determined for two tumours in each patient. The relative difference between the SUVs of these tumours (∆SUV) was determined and compared between the second primary group and metastatic disease group. Receiver-operating characteristic (ROC) curve analysis was performed to determine the sensitivity and specificity of the ∆SUV for an optimal cut-off value.ResultsA total of 37 patients (21 metastatic disease, 16 second primary cancer) were included for analysis. The ∆SUV was significantly higher in patients with second primary cancer than in those with metastatic disease (58 vs 28%, respectively, p < 0.001). The area under the ROC curve was 0.81 and the odds ratio for the optimal cut-off was 18.4.ConclusionSUVs from 18F-FDG PET images can be helpful in differentiating metastatic disease from second primary tumours in patients with synchronous pulmonary lesions. Further studies are warranted to confirm the consistency of these results.

Dichotomous ALK-IHC is a better predictor for ALK inhibition outcome than traditional ALK-FISH in advanced non-small cell lung cancer

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.

Mediastinal staging in daily practice: endosonography, followed by cervical mediastinoscopy. Do we really need both?

OBJECTIVES In patients with lung cancer, endosonography has emerged as a minimally invasive method to obtain cytological proof of mediastinal lymph nodes, suspicious for metastases on imaging. In case of a negative result, it is currently recommended that a cervical mediastinoscopy be performed additionally. However, in daily practice, a second procedure is often regarded superfluous. The goal of our study was to assess the additional value of a cervical mediastinoscopy, after a negative result of endosonography, in routine clinical practice. METHODS In a retrospective cohort study, the records of 147 consecutive patients with an indication for mediastinal lymph node staging and a negative result of endosonography were analysed. As a subsequent procedure, 124 patients underwent a cervical mediastinoscopy and 23 patients were scheduled for an intended curative resection directly. The negative predictive value (NPV) for both diagnostic procedures was determined, as well as the number of patients who needed to undergo a mediastinoscopy to find one false-negative result of endosonography (number needed to treat (NNT)). Clinical data of patients with a false-negative endosonography were analysed. RESULTS When using cervical mediastinoscopy as the gold standard, the NPV for endosonography was 88.7%, resulting in a NNT of 8.8 patients. For patients with fluoro-2-deoxyglucose positron emission tomography positive mediastinal lymph nodes, the NNT was 6.1. Overall, a futile thoracotomy could be prevented in 50% of patients by an additional mediastinoscopy. A representative lymph node aspirate, containing adequate numbers of lymphocytes, did not exclude metastases. CONCLUSIONS In patients with a high probability of mediastinal metastases, based on imaging, and negative endosonography, cervical mediastinoscopy should not be omitted, not even when the aspirate seems representative.

Irreversible Respiratory Failure in a Full-Term Infant with Features of Pulmonary Interstitial Glycogenosis as Well as Bronchopulmonary Dysplasia

Pulmonary interstitial glycogenosis (PIG) is a rare interstitial lung disease in the newborns. We report on the clinical presentation and pathological findings of a full-term male infant with pulmonary hypertension requiring extracorporeal membrane oxygenation (ECMO). An open lung biopsy demonstrated interstitial changes resembling pulmonary interstitial glycogenosis as well as bronchopulmonary dysplasia (BPD), without convincing evidence of maturational arrest, infection, alveolar proteinosis, or alveolar capillary dysplasia. The boy was treated with glucocorticoids and, after a few days, was weaned from ECMO. A few hours later, the patient died due to acute severe pulmonary hypertension with acute right ventricular failure. The etiology and underlying pathogenic mechanisms of PIG are unknown. The clinical outcomes are quite varied. Deaths have been reported when PIG exists with abnormal lung development and pulmonary vascular growth and congenital heart disease. No mortality has been reported in PIG together with BPD in full-term infants. In this article, we reported on a full-term infant with interstitial changes resembling PIG and BPD who expired despite no convincing evidence of an anatomical maturational arrest or congenital heart disease.

Influence of lung injury on cardiac output measurement using transpulmonary ultrasound dilution: a validation study in neonatal lambs

BACKGROUND Transpulmonary ultrasound dilution (TPUD) is a promising method for cardiac output (CO) measurement in severely ill neonates. The incidence of lung injury in this population is high, which might influence CO measurement using TPUD because of altered lung perfusion. We evaluated the influence of lung injury on the accuracy and precision of CO measurement using TPUD in an animal model. METHODS In nine neonatal lambs, central venous and arterial catheters were inserted and connected to the TPUD monitor. Repeated lavages with warmed isotonic saline were performed to gradually induce lung injury. CO measurements with TPUD (COtpud) were compared with those obtained by an ultrasonic transit-time flow probe around the main pulmonary artery (COufp). An increase in oxygenation index was used as an indicator of induced lung injury during the experiment. Post-mortem lung injury was confirmed by histopathological examination. RESULTS Fifty-five sessions of three paired CO measurements were analysed. The mean COufp was 1.53 litre min(-1) (range 0.66-2.35 litre min(-1)), and the mean COtpud was 1.65 litre min(-1) (range 0.78-2.91 litre min(-1)). The mean bias (standard deviation) between the two methods was 0.13 (0.15) litre min(-1) with limits of agreement of ±0.29 litre min(-1). The overall percentage error was 19.1%. The accuracy and precision did not change significantly during progressive lung injury. Histopathological severity scores were consistent with heterogeneous lung injury. The capability to track changes in CO using TPUD was moderate to good. CONCLUSIONS The accuracy and precision of CO measurement using TPUD is not influenced in the presence of heterogeneous lung injury in an animal model.

Mesenchymal cystic hamartoma? A revised diagnosis after 23 years

In 1994, we published a rare case report of a left sided pneumothorax caused by a mesenchymal cystic hamartoma (MCH) in this journal ( Thorax 1994;49:1175–6).1 After a follow-up of 23 years, the same patient was referred for a left sided pain and thoracic mass at age 36 years. At age 14 years, a left sided thoracotomy was performed for recurrent pneumothorax followed by lobectomy of the left lower lobe in which a MCH was diagnosed.1 Now his symptoms started almost 1 year before referral. Analysis showed an inhomogeneous mass in the lower left thoracic cavity with metabolic activity on the F-18-fluorodeoxyglucose positron emission tomography-PET scan. Follow up CT-scans showed slow progression (figure 1) and CT-guided biopsy showed a monophasic malignant tumour consisting of spindle cells, immunohistochemically positive for vimentin and epithelial membrane antigen (EMA) …