Gabrijela Kocjan

Age-dependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer

Polycomb group proteins (PCGs) are involved in repression of genes that are required for stem cell differentiation. Recently, it was shown that promoters of PCG target genes (PCGTs) are 12-fold more likely to be methylated in cancer than non-PCGTs. Age is the most important demographic risk factor for cancer, and we hypothesized that its carcinogenic potential may be referred by irreversibly stabilizing stem cell features. To test this, we analyzed the methylation status of over 27,000 CpGs mapping to promoters of approximately 14,000 genes in whole blood samples from 261 postmenopausal women. We demonstrate that stem cell PCGTs are far more likely to become methylated with age than non-targets (odds ratio = 5.3 [3.8-7.4], P < 10(-10)), independently of sex, tissue type, disease state, and methylation platform. We identified a specific subset of 69 PCGT CpGs that undergo hypermethylation with age and validated this methylation signature in seven independent data sets encompassing over 900 samples, including normal and cancer solid tissues and a population of bone marrow mesenchymal stem/stromal cells (P < 10(-5)). We find that the age-PCGT methylation signature is present in preneoplastic conditions and may drive gene expression changes associated with carcinogenesis. These findings shed substantial novel insights into the epigenetic effects of aging and support the view that age may predispose to malignant transformation by irreversibly stabilizing stem cell features.

Suitability of endobronchial ultrasound-guided transbronchial needle aspiration specimens for subtyping and genotyping of non-small cell lung cancer: a multicenter study of 774 patients.

RATIONALE The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and nonsquamous subtypes as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the subclassification and genotyping of NSCLC. OBJECTIVES To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC. METHODS Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across five centers in the United Kingdom between 2009 and 2011. MEASUREMENTS AND MAIN RESULTS The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% confidence interval [CI], 73-80). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted odds ratio, 0.50; 95% CI, 0.28-0.82; P = 0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value, and diagnostic accuracy of EBUS-TBNA in patients with NSCLC were 88% (95% CI, 86-91), 72% (95% CI, 66-77), and 91% (95% CI, 89-93), respectively. CONCLUSIONS This large, multicenter, pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for subtyping of NSCLC and EGFR mutation analysis and that the use of immunohistochemistry reduces the rate of NSCLC-NOS.

Endoscopic ultrasound-guided tissue sampling by combined fine needle aspiration and trucut needle biopsy: a prospective study.

Background and aims:  Endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) has a diagnostic accuracy of 70–90%, depending on the site under evaluation. In order to improve EUS‐guided tissue sampling a novel 19‐gauge trucut‐type needle has been designed to obtain core biopsies during EUS. We prospectively evaluated the safety and accuracy of EUS‐FNA alone versus combined EUS‐FNA and trucut needle biopsy (TNB) in patients referred to our Unit over a 3‐year period.

Suitability of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration Specimens for Subtyping and Genotyping of Non–Small Cell Lung Cancer

RATIONALE The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and nonsquamous subtypes as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the subclassification and genotyping of NSCLC. OBJECTIVES To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC. METHODS Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across five centers in the United Kingdom between 2009 and 2011. MEASUREMENTS AND MAIN RESULTS The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% confidence interval [CI], 73-80). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted odds ratio, 0.50; 95% CI, 0.28-0.82; P = 0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value, and diagnostic accuracy of EBUS-TBNA in patients with NSCLC were 88% (95% CI, 86-91), 72% (95% CI, 66-77), and 91% (95% CI, 89-93), respectively. CONCLUSIONS This large, multicenter, pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for subtyping of NSCLC and EGFR mutation analysis and that the use of immunohistochemistry reduces the rate of NSCLC-NOS.

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients

Background: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. Methods: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P jirovecii heat shock protein 70 (HSP70) gene to quantify P jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). Results: Sixty-one patients had 62 episodes of PCP (defined by detection of P jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range ∼13–18 608 copies/reaction; median ∼332) and was detectable but below the limit of quantification (∼5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range ∼6–590 copies/reaction; median ∼14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off >10 copies/reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). Conclusion: The HSP70 real-time PCR assay detects P jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P jirovecii DNA by real-time PCR may also discriminate between colonisation with P jirovecii and infection.

Combination of endobronchial ultrasound-guided transbronchial needle aspiration with standard bronchoscopic techniques for the diagnosis of stage I and stage II pulmonary sarcoidosis

Background and objective:  Standard bronchoscopic techniques (transbronchial lung biopsy and endobronchial biopsy) provide a diagnosis in 70% of patients with pulmonary sarcoidosis. Previous data suggest that endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) has a high sensitivity in patients with sarcoidosis. The feasibility and utility of combining EBUS‐TBNA with standard bronchoscopic techniques is unknown. The aim of this study was to evaluate the feasibility, safety and efficacy of combined EBUS‐TBNA and standard bronchoscopic techniques in patients with suspected sarcoidosis and enlarged mediastinal or hilar lymphadenopathy.

The Interobserver Reproducibility of Thyroid Fine-Needle Aspiration Using the UK Royal College of Pathologists’ Classification System

The overall interobserver reproducibility of thyroid fine-needle aspiration (FNA) has not been comprehensively assessed. A blinded 6-rater interobserver reproducibility study was conducted of 200 thyroid FNA cases using the UK System, which is similar to The Bethesda System for Reporting Thyroid Cytology: Thy1, nondiagnostic; Thy2, nonneoplastic; Thy3a, atypia, probably benign; Thy3f, follicular lesion; Thy4, suspicious of malignancy; and Thy5, malignant. There was good interobserver agreement for the Thy1 (κ = 0.69) and Thy5 (κ = 0.61), moderate agreement for Thy2 (κ = 0.55) and Thy3f (κ = 0.51), and poor agreement for Thy3a (κ = 0.11) and Thy4 (κ = 0.17) categories. Combining categories implying surgical management (Thy3f, Thy4, and Thy5) achieved good agreement (κ = 0.72), as did combining categories implying medical management (Thy1, Thy2, and Thy3a; κ = 0.72). The UK thyroid FNA terminology is a reproducible and clinically relevant system for thyroid FNA reporting. This study demonstrates that international efforts to harmonize and refine thyroid cytology classification systems can improve consistency in the clinical management of thyroid nodules.

The role of breast FNAC in diagnosis and clinical management: a survey of current practice.

Most participating countries have now adopted a triple assessment approach, i.e. clinical,imaging and pathology, to breast diagnosis, with FNAC as the first‐line pathological investigation in both screening and symptomatic populations, with the exception of microcalcifications. Pathologists specialized in cytopathology are best qualified to collect and interpret FNAC samples, but this is not always possible or practical. Radiologists involved in breast imaging should ensure that they have the necessary skills to carry out FNAC under all forms of image guidance. Best results are achieved by a combination of both techniques, as shown in the image‐guided FNAC in the presence of the cytopathologist. The majority of European countries use similar reporting systems for breast FNAC (C1–C5), in keeping with European Guidelines for Quality Assurance in Breast Cancer Screening and Diagnosis, although some still prefer descriptive reporting only. When triple assessment is concordant, final treatment may proceed on the basis of FNAC, without a tissue biopsy. ER and PR assessment can be done safely on FNAC material. However, not all institutions may have expertise in doing this. HER‐2 protein expression on direct cytological preparations is insufficiently reliable for clinical use, although its use for FISH is possible, if expertise is available. The majority of participants practise a degree of one‐stop diagnosis with a cytopathologist present in the out‐patient clinic. Formal recognition of the importance of the time spent outside the laboratory, both for cytopathologist and cytotechnologist, is necessary in order to ensure appropriate resourcing. The use of core biopsy (CB) has increased, although not always for evidence‐based reasons. CB and FNAC are not mutually exclusive. FNAC should be used in diagnosis of benign, symptomatic lesions and CB in microcalcifications, suspicious FNAC findings and malignancies where radiology cannot guarantee stromal invasion.

BSCC Code of Practice--fine needle aspiration cytology.

The British Society for Clinical Cytology Code of Practice on fine needle aspiration cytology complements that on exfoliative cytopathology, which was published in the last issue (Cytopathology 2009;20:211–23). Both have been prepared with wide consultation within and outside the BSCC and have been endorsed by the Royal College of Pathologists. A separate code of practice for gynaecological cytopathology is in preparation. Fine needle aspiration (FNA) cytology is an accepted first line investigation for mass lesions, which may be targeted by palpation or a variety of imaging methods. Although FNA cytology has been shown to be a cost‐effective, reliable technique its accurate interpretation depends on obtaining adequately cellular samples prepared to a high standard. Its accuracy and cost‐effectiveness can be seriously compromised by inadequate samples. Although cytopathologists, radiologists, nurses or clinicians may take FNAs, they must be adequately trained, experienced and subject to regular audit. The best results are obtained when a pathologist or an experienced and trained biomedical scientist (cytotechnologist) provides immediate on‐site assessment of sample adequacy whether or not the FNA requires image‐guidance. This COP provides evidence‐based recommendations for setting up FNA services, managing the patients, taking the samples, preparing the slides, collecting material for ancillary tests, providing rapid on‐site assessment, classifying the diagnosis and providing a final report. Costs, cost‐effectiveness and rare complications are taken into account as well as the time and resources required for quality control, audit and correlation of cytology with histology and outcome. Laboratories are expected to have an effective quality management system conforming to the requirements of a recognised accreditation scheme such as Clinical Pathology Accreditation (UK) Ltd.

Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration for the Diagnosis of Intrathoracic Lymphadenopathy in Patients with Extrathoracic Malignancy: A Multicenter Study

Introduction: Mediastinal lymphadenopathy in patients with an extrathoracic malignancy is a common clinical scenario. Invasive sampling of intrathoracic lymph nodes may be performed by mediastinoscopy or endoscopic ultrasound-guided fine needle aspiration. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an alternative to mediastinoscopy and endoscopic ultrasound in patients with lung cancer and sarcoidosis. The utility of EBUS-TBNA in patients with extrathoracic malignancy was evaluated. Methods: Consecutive patients who were suspected to have intrathoracic lymph node metastases from an extrathoracic malignancy underwent EBUS-TBNA. When EBUS-TBNA did not provide a specific diagnosis, patients underwent mediastinoscopy or clinical follow-up of at least 6 months duration. Results: One hundred sixty-one patients meeting the inclusion criteria underwent EBUS-TBNA in five UK centers over a 3-year period. EBUS-TBNA diagnosed mediastinal or hilar metastases in 71 (44%) patients, new lung cancer in 20 (12%) patients, and sarcoidosis in 14 (9%) patients. The sensitivity, negative predictive value for malignancy, and overall accuracy for EBUS-TBNA were 87%, 73% and 88%, respectively. One hundred ten (68%) patients in the study had a final diagnosis of malignant intrathoracic lymphadenopathy. Conclusion: Because of the high prevalence of alternative diagnoses, pathological evaluation is important in patients with extrathoracic malignancy and suspected mediastinal or hilar lymph node metastases. EBUS-TBNA is a safe and sensitive technique and may be considered a first-line investigation in these patients.