Camilla E. Comin

Design, recruitment and baseline results of the ITALUNG trial for lung cancer screening with low-dose CT

BACKGROUND Results of randomized clinical trials (RCTs) are needed to assess the efficacy of lung cancer screening with low-dose chest computed tomography (CT) in reducing lung cancer mortality. We report design and results of enrolment and baseline screening test in the ITALUNG trial, a RCT. METHODS Invitation letters were sent to subjects of 55-69 years of age clients of 269 general practitioners. Smokers or former smokers of at least 20 pack/years were eligible and after written consent were randomized in an active arm undergoing a low-dose CT annually for 4 years and in a control arm receiving no screening. Management of positive screening test was carried out using follow-up low-dose CT, fluorodeoxyglucose positron emission tomography, fine needle aspiration cytology and fiber optic bronchoscopy. RESULTS A sample of 3206 eligible subjects was achieved by sending 71,232 letters (enrolment efficacy = 4.5%). Subjects in control (n = 1593) and active (n = 1613) arm were balanced for age, gender and smoking history. Two-hundred and seven (12.8%) subjects did not undergo CT after randomization. The baseline screening test was positive in 426 (30.3%) of 1406 subjects. Twenty-one lung cancers (prevalence = 1.5%) were found in 20 subjects: 18 non-small cell lung cancer (NSCLC), 2 small cell lung cancer (SCLC) and a case of typical carcinoid. Ten NSCLC (47.6%) were in Stage I. Sixteen fine needle aspirations were performed in 15 lung cancers, with a positive result in 12 (75%) cases. One biopsy only (6.3%) was performed on a benign lesion. Seventeen lung cancers (81%) were treated with surgical resection in 16 subjects. One subject underwent surgery for a benign lesion (5.5% of all surgical resections). CONCLUSIONS Recruitment by mail of high risk subjects for a lung cancer screening RCT is feasible but not efficient. Results of the baseline screening test in the active arm of the ITALUNG trial are substantially in line with those of RCT and observational studies.

The first tissue-engineered airway transplantation: 5-year follow-up results

BACKGROUND In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results. METHODS The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen. FINDINGS By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life. INTERPRETATION These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.

h-caldesmon, Calretinin, Estrogen Receptor, and Ber-ep4: A Useful Combination of Immunohistochemical Markers for Differentiating Epithelioid Peritoneal Mesothelioma From Serous Papillary Carcinoma of the Ovary

Distinguishing between epithelioid peritoneal mesothelioma and papillary serous carcinomas involving the peritoneum may be very difficult, owing to overlapping morphologic features. Immunohistochemistry may facilitate establishing a correct diagnosis, but, as no single antibody has demonstrated absolute sensitivity and specificity for either mesothelioma or serous carcinoma, the differential diagnosis is based mainly on the combined use of several markers. The purpose of this study was to ascertain the sensitivity and specificity of a series of mesothelial markers [including more recently investigated antigens such as h-caldesmon (h-CD) and D2-40] and, using receiver operating characteristic curve analysis, to identify a selected appropriate panel of antibodies for differentiating between epithelioid peritoneal mesothelioma and serous papillary carcinoma of the ovary. Fifteen cases of epithelioid peritoneal mesothelioma and 40 cases of papillary serous carcinoma of the ovary (25 primary and 15 metastatic to the peritoneum) were immunostained for h-CD, D2-40, calretinin, cytokeratin 5/6, thrombomodulin, estrogen and progesterone receptors (ER and PR), Ber-EP4, B72.3, CA19-9, and CD15. h-CD and calretinin showed the highest sensitivity (100%), followed by D2-40 (93.3%) and cytokeratin 5/6 (93.3%); thrombomodulin had the lowest sensitivity (60%). h-CD and thrombomodulin had the best specificity (95%) for mesothelioma, followed by calretinin (87.5%), D2-40 (80%), and cytokeratin 5/6 (72.5%). Among carcinoma markers, ER and Ber-EP4 demonstrated the highest sensitivity (95%) followed by B72.3 (72.5%), PR (65%), CA19.9 (60%), and CD15 (45%). The specificity of the nonmesothelial markers was 100%, except for Ber-EP4 (93.3%). The relationship between the values of sensitivity and specificity of each marker using receiver operating characteristic analysis permitted the identification of h-CD, calretinin, ER, and Ber-EP4 as the markers with the best performance in differentiating epithelioid peritoneal mesothelioma from serous papillary carcinoma of the ovary.

Development of bioengineered human larynx.

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.

Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

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.

Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue

In this study, the obtainment and characterization of decellularized rat tracheal grafts are described. The detergent-enzymatic method, already used to develop bioengineered pig and human trachea scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be used to improve our knowledge on the process of tissue-engineered airway transplantation and regeneration. The results demonstrated that, after 9 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues with strong in vivo angiogenic characteristics, could be obtained. Moreover, to improve the mechanical properties of decellularized tracheae, genipin is here considered as a naturally derived cross-linking agent. The results demonstrated that the treatment increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response.

Microvessel density and clinicopathological characteristics in hepatitis C virus and hepatitis B virus related hepatocellular carcinoma

Aims: To compare intratumorous microvessel density (MVD) and clinicopathological features in two different groups of hepatocellular carcinoma (HCC), namely: hepatitis B virus (HBV) related HCC (B-HCC) and HCV related HCC (C-HCC). Methods: Fifty consecutive cases each of B-HCC and of C-HCC were studied. Microvessel numbers were assessed by staining for the antigen CD34; in each case, three areas with the highest numbers of microvessels were counted in both the intratumorous and the surrounding non-tumorous tissue; the mean value represented the final MVD. Results: Patients with B-HCC were significantly younger than those with C-HCC (mean age, 60.1 (SD, 4.1) v 66.4 (4.3) years); no significant differences were seen for sex or Child’s class distribution. The tumour diameter was larger in B-HCCs than in C-HCCs (mean, 5.6 (SD, 1.8) v 3.8 (1.8) cm). Tumour microsatellite formation was significantly higher in C-HCCs (12 v 4 cases). No differences were found for histological subtype, degree of differentiation, tumour encapsulation, and vascular invasion. The mean MVD value was significantly higher in tumorous (mean, 54 (SD, 13.8) v 38 (8.9)) and in the surrounding non-tumorous liver tissue (mean, 15 (SD, 4.3) v 7 (3.1)) of C-HCCs. Conclusions: C-HCCs present as smaller tumours in older patients, with a higher incidence of tumour microsatellite formation and higher MVD values both in the tumorous and the non-tumorous areas, suggesting a link between HCV infection, angiogenesis, and hepatocarcinogenesis.

Clear cell sarcoma of the ileum: report of a case and review of literature

Clear cell sarcoma (CCS) is a high grade soft tissue sarcoma with a distinct molecular profile and with morphological features resembling those of melanoma. CCS has been rarely described in other locations other than the soft tissues, including the gastrointestinal tract. In this study, we report a case of CCS arising in the ileum of a 31-year-old woman. Histologically, the tumor involved the entire thickness of the intestinal wall. Tumor cells were polygonal or fusiform, with clear or eosinophilic cytoplasm, arranged in a uniform nested to fascicular growth pattern. Immunohistochemical studies revealed strong positivity for vimentin and S-100 protein. HMB-45, Melan-A, tyrosinase, cytokeratins, EMA, smooth muscle actin, CD34, CD31, CD117, CD99, synaptophysin, chromogranin A, CD56, and NSE were negative. Fluorescence in situ hybridization analysis demonstrated the presence of a t(12;22)(q13;q12) translocation, the diagnostic hallmark of CCS of soft parts. The present case, together with a detailed review of the literature on this topic, demonstrates that the gastrointestinal tract is a possible site of CCS of soft tissues and that making a reliable diagnosis of this tumor requires cytogenetic or molecular diagnostic investigations.

hERG1 channels in human esophagus: evidence for their aberrant expression in the malignant progression of Barrett's esophagus.

Ion channels regulate a broad range of cellular activities. Alteration in ion channel function has been reported in different human pathologies, such as cardiac, neuromuscular, autoimmune diseases, and cancer. We investigated the expression of hERG1 K+ channels in the human upper gastrointestinal tract, focusing our attention on the lower esophagus. In particular, we analyzed by both Reverse transcription and polymerase chain reaction (RT‐PCR) and immunohistochemistry (IHC) endoscopic samples obtained from normal subjects, from patients suffering from gastroesophageal reflux, associated or not with esophagitis, and from patients affected by Barretts esophagus (BE), that is, intestinal metaplasia. None of the normal samples, nor those from patients with gastro‐esophageal reflux symptoms and reflux esophagitis expressed the hERG1 protein. On the other hand, 69% of patients with BE expressed hERG1. Since BE is a preneoplastic lesion, dysplasias (Ds) and adenocarcinomas (ADKs) arising on a previously diagnosed BE were also analyzed, and all the samples showed a high expression of the hERG1 protein. The surveillance of patients with BE showed that 89% of those who later developed ADKs displayed hERG1 expression. Data here reported, support the hypothesis that hERG1 expression marks an early step of the progression of normality to cancer in the human esophagus through a metaplastic and dysplastic stage. J. Cell. Physiol. 209: 398–404, 2006.