Competence in flexible bronchoscopy and basic biopsy technique.

Diagnostic bronchoscopy and tissue sampling techniques using forceps (endobronchial biopsy [EBB] and transbronchial biopsies [TBB]) or needle aspiration (transbronchial needle aspiration-TBNA), all performed with a flexible bronchoscope, are the basic elements of any interventional procedure. The flexible fibrobronchoscopy allows the visualization of the airways and is used both for diagnostic and therapeutic purposes. The working channel of both fibrobronchoscopes with optical fibers and videobronchoscopes, even if of relatively small diameter, allows the insertion of various diagnostic and therapeutic accessories. Fiber optic systems have been widely replaced by video cameras using a miniaturized charge-coupled device camera positioned at the end of the scope that provides electronic transmission of images to a monitor. The indications for both diagnostic and therapeutic fibrobronchoscopy derive from a correct evaluation of symptoms and objective signs of the patient and from the correct interpretation of imaging methods. Although bronchoscopy techniques keep evolving at a rapid pace, basic procedures such as bronchoalveolar lavage, transbronchial lung biopsy, and transbronchial needle aspiration still play a key role in pulmonary disease diagnostics, and therefore, these methods must still be part of the training of interventional pulmonologists. Trainees will acquire a thorough knowledge of thoracic anatomy and become skilled in the interpretation of thoracic imaging, after which they will be given a theoretical and practical training course on virtual reality simulators, on animal or cadaver models, the effectiveness of which has been fully demonstrated by scientific studies. Specific DOPS tests have been developed for a qualitative evaluation of procedures on simulators, on animal models and on the patient.

Screen-detected multiple primary lung cancers in the ITALUNG trial.

Occurrence of multiple primary lung cancers (MPLC) in individuals undergoing low-dose computed tomography (LDCT) screening has not been thoroughly addressed. We investigated MPLC in subjects recruited in the ITALUNG randomized clinical trial. Cases of cytologically/histologically proven MPLC detected at screening LDCT or follow-up CT were selected and pathologically re-evaluated according to the WHO 2015 classification. Overall 16 MPLC were diagnosed at screening LDCT (n=14, all present at baseline) or follow-up CT (n=2) in six subjects (4 in one subject, 3 in two and 2 in three subjects), representing 0.43% of the 1,406 screenees and 15.8% of the 38 subjects with at least one screen-detected primary lung cancer. MPLC included 9 adenocarcinomas in three subjects and a combination of 7 different tumour histotypes in three subjects. MPLC, mostly adenocarcinomas, are not uncommon in smokers and ex-smokers with at least one LDCT screen detected primary lung cancer.

Circulating tumor cells and microemboli can differentiate malignant and benign pulmonary lesions.

The presence of circulating tumor cells (CTC) or microemboli (CTM) in the peripheral blood can theoretically anticipate malignancy of solid lesions in a variety of organs. We aimed to preliminarily assess this capability in patients with pulmonary lesions of suspected malignant nature. We used a cell-size filtration method (ScreenCell) and cytomorphometric criteria to detect CTC/CTM in a 3 mL sample of peripheral blood that was taken just before diagnostic percutaneous CT-guided fine needle aspiration (FNA) or core biopsy of the suspicious lung lesion. At least one CTC/CTM was found in 47 of 67 (70%) patients with final diagnoses of lung malignancy and in none of 8 patients with benign pulmonary nodules. In particular they were detected in 38 (69%) of 55 primary lung cancers and in 9 (75%) of 12 lung metastases from extra-pulmonary cancers. Sensitivity of CTC/CTM presence for malignancy was 70.1% (95%CI: 56.9-83.1%), specificity 100%, positive predictive value 100% and negative predictive value 28.6% (95%CI: 11.9-45.3%). Remarkably, the presence of CTC/CTM anticipated the diagnosis of primary lung cancer in 3 of 5 patients with non-diagnostic or inconclusive results of FNA or core biopsy, whereas CTC/CTM were not observed in 1 patient with sarcoidosis and 1 with amarthocondroma. These results suggest that presently, due to the low sensitivity, the search of CTC/CTM cannot replace CT guided percutaneous FNA or core biopsy in the diagnostic work-up of patients with suspicious malignant lung lesions. However, the high specificity may as yet indicate a role in cases with non-diagnostic or inconclusive FNA or core biopsy results that warrants to be further investigated.

Prevalence and number of circulating tumour cells and microemboli at diagnosis of advanced NSCLC.

PURPOSE: Timing and magnitude of blood release of circulating tumour cells (CTC) and circulating tumour microemboli (CTM) from primary solid cancers are uncertain. We investigated prevalence and number of CTC and CTM at diagnosis of advanced non-small cell lung cancer (NSCLC). METHODS: Twenty-eight consecutive patients with suspected stage III-IV lung cancer gave consent to provide 15 mL of peripheral blood soon before diagnostic CT-guided fine-needle aspiration biopsy (FNAB). CTC and CTM (clusters of ≥3 CTC) were isolated by cell size filtration (ScreenCell), identified and counted by cytopathologists using morphometric criteria and (in 6 cases) immunostained for vimentin. RESULTS: FNAB demonstrated NSCLC in 26 cases. At least one CTC/3 mL blood (mean 6.8 ± 3.7) was detected in 17 (65 %) and one CTM (mean 4.5 ± 3.3) in 15 (58 %) of 26 NSCLC cases. No correlation between number of CTC or CTM and tumour type or stage was observed. Neoplastic cells from both FNA and CTC/CTM were positive for vimentin but heterogeneously. CONCLUSIONS: CTC can be detected in two-thirds and CTM in more than half of patients with advanced NSCLC at diagnosis. Reasons underlying lack of CTC and CTM in some advanced lung cancers deserve further investigations.

ALK and ROS1 rearrangements tested by fluorescence in situ hybridization in cytological smears from advanced non-small cell lung cancer patients.

BACKGROUND: The identification of ALK and ROS1 rearrangements and the availability of an effective target therapy, such as crizotinib, represent a new option in the treatment of advanced non-small cell lung cancer (NSCLC) patients. In light of recent advances in non-invasive diagnostic procedures, we aimed to demonstrate that direct cytological smears are suitable for assessing ALK and ROS1 rearrangements in patients with NSCLC. METHODS: Fifty-five patients with a cytological diagnosis of lung adenocarcinoma (ADC) were evaluated for ALK rearrangements by fluorescence in situ hybridization (FISH) and 12 patients for ROS1 FISH rearrangements. Seventeen of the 55 cytological samples tested for ALK were obtained from the primary tumor and 38 from metastatic lesions. Ten of 12 samples evaluated for ROS1 were obtained from metastatic sites and two from the primary tumor. RESULTS: ALK FISH was successful in 49/55 (89%) cytological ADC samples and ROS1 FISH in all 12 cytological samples. ALK rearrangements were found in 3/13 (23%) primary tumors and 7/36 (19%) metastatic sites. ROS1 rearrangements were found in one of the two primary tumors and in two of the 10 metastases. Two of the three rearranged cases were tested on cytology after knowing that they were rearranged on histology in order to increase representativeness of ROS1 rearranged cases in this study. CONCLUSION: Whenever cytology represents the only available material for diagnosis and biological characterization of NSCLC, minimally invasive procedures may provide an additional important source of cellular material for FISH assessment of ALK and ROS1 rearrangements.

Four-year results of low-dose CT screening and nodule management in the ITALUNG trial.

INTRODUCTION: Recruitment and nodule management are critical issues of lung cancer screening with low-dose computed tomography (LDCT). We report subjects' compliance and results of LDCT screening and management protocol in the active arm of the ITALUNG trial. METHODS: Three thousand two hundred six smokers or former smokers invited by mail were randomized to receive four annual LDCT (n = 1613) or usual care (n = 1593). Management protocol included follow-up LDCT, 2-[18F]fluoro-2-deoxy-D glucose positron emission tomography (FDG-PET), and CT-guided fine-needle aspiration biopsy (FNAB). RESULTS: One thousand four hundred six subjects (87%) underwent baseline LDCT, and 1263 (79%) completed four screening rounds. LDCT was positive in 30.3% of the subjects at baseline and 15.8% subsequently. Twenty-one lung tumors in 20 subjects (1.5% detection) were found at baseline, and 20 lung tumors in 18 subjects (0.5% detection) in subsequent screening rounds. Ten of 18 prevalent (55%) and 13 of 17 incident (76%) non-small-cell cancers were in stage I. Interval growth enabled diagnosis of lung cancer in 16 subjects (42%), but at least one follow-up LDCT was obtained in 741 subjects (52.7%) over the screening period. FDG-PET obtained in 6.5% of subjects had 84% sensitivity and 90% specificity for malignant lesions. FNAB obtained in 2.4% of subjects showed 90% sensitivity and 88% specificity. Positivity of both FDG-PET and FNAB invariably predicted malignancy. Surgery for benign lesions was performed on four subjects (10% of procedures) but followed protocol violations on three subjects. CONCLUSIONS: High-risk subjects recruited by mail who entered LDCT screening showed a high and stable compliance. Efficacy of screening is, however, weakened by low detection rate and specificity. Adhesion to management protocol might lessen surgery for benign lesions.

From clinical trials to clinical practice: predictors of response to erlotinib in advanced non-small cell lung cancer patients pretreated with chemotherapy.

BACKGROUND: Inhibition of the epidermal growth factor receptor pathway with tyrosine kinase inhibitors can improve outcome of patients with advanced non-small cell lung cancer after first-line chemotherapy. The use of clinical characteristics and molecular markers may permit the identification of patients who are more likely to benefit from erlotinib. PATIENTS AND METHODS: Retrospective analysis of unselected patients with metastatic non-small cell lung cancer who had previously failed on at least one line of chemotherapy and treated at our institution with erlotinib (150 mg/day orally) until disease progression. Mutations of epidermal growth factor receptor (exon 19-21) and KRAS (codon 12-13) genes were screened with high-resolution melting analysis and identified with direct sequencing. RESULTS: Fifty-three patients were included in the study. The disease control rate was 38%. Median progression-free survival and median overall survival were 4 and 15 months, respectively. Skin rash, diarrhea and mucositis were the most common toxicities of erlotinib. In 19 patients, erlotinib dose was reduced for toxicity. The disease control rate and progression-free survival were significantly better in non-smokers, responders to chemotherapy and patients with epidermal growth factor receptor mutations. Overall survival was longer in patients with skin toxicity and epidermal growth factor receptor mutations. CONCLUSIONS: In our experience, epidermal growth factor receptor mutations, response to previous chemotherapy and non-smoking status were predictors of higher disease control rate and longer progression-free survival. Overall survival was significantly longer in patients with epidermal growth factor receptor mutations and skin toxicity.

Epidermal growth factor receptor and K-RAS mutations in 411 lung adenocarcinoma: a population-based prospective study.

Targeting the epidermal growth factor receptor has played a central role in advanced non-small cell lung cancer research, treatment, and patient outcomes over the last several years; however, a number of questions about this approach remain to be addressed. Through the Istituto Toscano Tumori and the Italian Association of Women Against Lung Cancer Project, we collected 411 lung adenocarcinomas from several clinical centers in Tuscany. Mutations were assessed by sequencing exons 18-21 of the epidermal growth factor receptor gene, and by restriction fragment length polymorphism analysis of codons 12 and 13 of the K-RAS gene. Epidermal growth factor receptor mutations (12.6%) were more frequently observed in females (p<0.0001), in non-smokers (p=0.005), and in the presence of bronchioloalveolar features (p=0.0004). K-RAS mutations (17.9%) were more frequent in males (p=0.0007) and were associated with smoking habits (p=0.005). Epidermal growth factor receptor and K-RAS mutations were mutually exclusive (p=0.001). We focused on 21 female patients with advanced/metastatic lung adenocarcinoma who received gefitinib 250 mg/day (expanded access) or erlotinib 150 mg/die as second/third-line therapy; partial response was associated with classic epidermal growth factor receptor mutations (p=0.006) and with a non-smoking history (p=0.02). None of the female patients with partial response and/or stable disease showed K-RAS alterations. Although the data obtained in our study have yet to be analyzed and confirmed with a larger number of patients treated with tyrosine kinase inhibitors, they should provide useful information for targeted therapy, in particular for non-smoking female lung cancer patients.

Accuracy of fine needle aspiration cytology (FNAC) of axillary lymph nodes as a triage test in breast cancer staging.

INTRODUCTION: Axillary node fine needle aspiration cytology (FNAC) has the potential to triage women with operable breast cancer to initial nodal surgical procedure. Because of variability in the reported accuracy of this test its role and clinical utility in pre-operative staging remains controversial. METHODS: We retrospectively evaluated the accuracy of ultrasound-guided axillary FNAC in all consecutive clinically T1-2 N0-1 breast cancers that had undergone this test (491 biopsies). We included subjects with clinically or sonographically indeterminate or suspicious nodes. Pathological node status was used as the reference standard (based on axillary dissection or sentinel node biopsy). RESULTS: Sensitivity of node FNAC was 72.6% (67.3-77.9) and specificity was 95.7% (92.5-98.8) for all cases, sensitivity was lower at 64.6% (59.3-70.0) if inadequate cytology was included as a negative result. FNAC sensitivity was highest in women with clinically suspicious nodes [92.5% (88.2-96.7)] and lowest in women with sonographically abnormal and clinically negative nodes [50.0% (41.3-58.7)]. Specificity was high in both groups, 81.2% (54.5-96.0) and 97.2% (94.6-99.9), respectively. The false-negative rate was 15.3% (12.1-18.5), the false-positive rate was 1.4% (0.4-2.5), and the inadequacy rate was 10.8% (8.0-13.5). The likelihood of node FNAC being positive was significantly associated with tumour grade and stage, and the number of nodes involved with metastases. DISCUSSION: Our data show that axillary FNAC has moderate sensitivity (which varies according to selection criteria for the test) and consistently high specificity, is associated with low inadequacy and very few false positives. We estimate that its use would have improved triage to initial nodal procedure in about one quarter of our cases. If one accepts the premise that initial surgical staging of the axilla should be based on all information available through pre-operative diagnosis, then axillary FNAC should be adopted routinely into clinical practice.

Prognostic significance of p53 and Ki-67 antigen expression in surgically treated non-small cell lung cancer: immunocytochemical detection with imprint cytology.

The purpose of this study was to determine the prognostic significance of the expression of p53 and Ki-67 in non-small cell lung cancer (NSCLC) using immunocytochemical detection. All consecutive NSCLC cases were selected for study, and, after surgery, a part of each tumor sample was frozen at -20 degrees C and stored for immunocytochemical studies. Overexpression of p53 was associated significantly with worse patient outcome in stage I disease, whereas no excess risk was evident in stage II and III cases. The same pattern was observed for Ki-67 expression. The excess risk in stage I cases with p53 and Ki-67 overexpression was observed only in adenocarcinoma. These findings are in agreement with other retrospective studies and support the hypothesis that p53 alteration may have different roles in adenocarcinoma and in squamous cell carcinoma, such as a carcinogenic factor for both cellular types but progression only for adenocarcinoma.

On-site evaluation of percutaneous CT-guided fine needle aspiration of pulmonary lesions. A study of 321 cases.

PURPOSE: The aim of this study was to assess the usefulness of having a cytopathologist present during percutaneous CT-guided fine needle aspiration (FNA) of pulmonary lesions. MATERIALS AND METHODS: Three hundred and twenty-one FNAs of lung lesions were performed in 312 patients (218 males, 94 females; age range: 20-86 years; mean age: 66 yrs). Nodule sizes ranged from 0.5 to 8 cm. The sampling was performed by a radiologist under CT-guidance; the calibre of the needle used was 20-25 G. Smears were prepared in the Radiology Department and stained using a quick method by a cytopathologist: the sample adequacy was assessed and, if possible, a preliminary diagnosis was made. An additional FNA was requested if the first aspirate was considered diagnostically inadequate. The diagnostic accuracy was examined by reviewing the clinical data and by correlating with the histological material and the clinical outcome. RESULTS: Satisfactory diagnostic material was obtained in 275 of the 321 FNA (86%): 231 were malignant (72%), 8 suspicious (3%) and 36 were negative for malignancy (11%). Forty-six of the 321 specimens (14%) were considered inadequate for any diagnostic verification. An additional FNA was performed immediately in 60 cases (19%). Forty-two subjects developed pneumothorax: 21 patients (50%) of these required thoracic drainage. Blood effusion around the lesion site or adjacent to the needle path was observed in 39 patients (12%). Sensitivity was 99%, specificity was 95% and diagnostic accuracy was 99%. CONCLUSIONS: CT guided aspiration cytology can be a safe and fast procedure for lung nodule characterisation. On-site immediate evaluation of FNA specimens can be beneficial in determining the adequacy of the aspirate and in providing accurate preliminary diagnoses of the specimens, thus allowing for rapid clinical decisions.

Analysis of morphologic patterns of fine-needle aspiration of the breast to reduce false-negative results in breast cytology.

BACKGROUND: The identification of specific morphologic diagnostic criteria is of paramount importance to optimize the accuracy of fine-needle aspiration cytology (FNAC) and to reduce the rate of false-negative results. In the current study, the authors reviewed a consecutive series of false-negative findings observed in the study center to define the presence and degree of cytologic abnormalities. False-negative cases were randomly mixed with true-negative cases and were reviewed by a panel of expert readers in a blinded fashion. The main objective of the current study was to identify a morphologic pattern that may permit the reduction of false-negative findings while maintaining the specificity of FNAC. METHODS: A blind review of a set of 41 consecutive false-negative and 49 true-negative breast aspiration samples was performed by a panel of 10 expert cytologists who were asked to give a final report and to classify the samples according to classic morphologic parameters. RESULTS: The majority final report sensitivity was 54% (range, 19-61%) and specificity was 73% (range, 65-92%). The average concordance with the majority report, adjusted for chance agreement (kappa statistic), was moderate at 0.54 (range, 0.40-0.65). Enlarged nuclear size, a hyperchromatic nucleus, the absence of naked nuclei, and the absence of apocrine metaplasia were reported more frequently in carcinoma cases, although not to a significant extent. The only variable found to be associated significantly (P = 0.041) with a diagnosis of carcinoma was the presence of microcalcifications, which nevertheless were found to occur in only a minority of carcinoma cases (7 of 41 cases) or controls (2 of 49 controls). Multivariate analysis demonstrated that the presence of microcalcifications (odds ration [OR] of 3.0; 95% confidence interval [95% CI], 1.2-7.4), the absence of naked nuclei (OR of 2.4; 95% CI, 1.3-4.4), and enlargement of the nucleus (OR of 1.9; 95% CI, 1.1-3.4) were all independently associated with false-negative findings. Diagnostic accuracy using a morphology-based score did not appear to improve the results substantially compared with the final report (sensitivity of 0.46 vs. 0.54 [P = 0.508] and a specificity of 0.80 vs. 0.73 [P = 0.218]). CONCLUSIONS: The results of the current study confirm that breast FNAC false-negative results are at least partially the result of underreporting of abnormalities that may be noted at review. Detailed analysis of a single morphologic characteristic was found to be of limited diagnostic value, suggesting that operators do perceive abnormalities but cannot translate these findings into distinct morphologic categories.

A feasibility study of the use of the AutoPap screening system as a primary screening and location-guided rescreening device.

BACKGROUND: The AutoPap 300 QC system (Tripath Imaging, Inc., Burlington, NC) is an automated device that was designed to screen conventionally prepared cervical smears and, more recently, thin-layer slide preparations. The system has been tested in large clinical trials. METHODS: A total of 14,145 cervical smears obtained from participants in the Florence screening program were eligible for the study. Smears were processed first with the AutoPap system and were classified into three different categories: 1) no further review (NFR), 2) review, and 3) process review (PR). Conventional manual reading was performed by 10 experienced cytopathologists. RESULTS: After AutoPap processing, 2398 smears were classified as NFR (16.9%), and 1818 smears were classified as PR (12.8%). Overall, there were 188 inadequate smears (1.3%) at conventional review and 125 inadequate smears (0.88%) at AutoPap review. Six-month repeat smears were prompted by 330 conventional reviews (2.3%) and by 222 AutoPap reviews (1.56%). Similarly, referral to colposcopy was prompted by 179 conventional reviews (1.2%) and by 147 AutoPap reviews (1.0%). Overall, 32 patients were diagnosed with high-grade cervical intraepithelial neoplasia as a result of assessment. Conventional reading detected 31 patients (28 patients were referred for colposcopy, and 3 patients were referred for repeat cytology), and the AutoPap system detected 30 patients (27 patients were referred for colposcopy, and 3 patients were referred for repeat cytology). CONCLUSIONS: The current experience suggested that conventional reading and AutoPap reading of cervical smears had essentially the same sensitivity, with slightly greater specificity for the AutoPap system. Thus, comparisons of the AutoPap system and conventional reading should focus mainly on cost analysis.

Conventional pap smear and liquid-based cervical cytology smear: comparison from the same patient.

BACKGROUND: The results of blind reading of smears obtained with liquid-based cytology in patients previously screened by conventional cytology were compared. MATERIAL AND METHODS: Cases selected for the study were a consecutive series of 99 subjects undergoing colposcopy within the screening program of the Florence District. The Pap test samples were processed utilizing the Thin Prep 2000 (Cytyc Corporation, Boxborough, MA). The liquid-base cytology smears were randomly admixed and read by seven expert cytologists with more than 15 years of experience in Pap smear reading. For each case, a consensus diagnosis was created and considered as the definitive diagnosis. Cytologic reports in conventional and liquid-based cytology smears were compared by the kappa statistic to evaluate diagnostic agreement. RESULTS: The study showed that the conventional and liquid-based cytology provide comparable cytologic reports and that the latter is not less sensitive than the former in detecting CIN2+ lesions of the cervix. DISCUSSION: Such evidence suggests the feasibility of randomized studies comparing the two methods, which are needed before adopting liquid-based cytology as the current method when screening for cervical cancer.