Lung ultrasound compared to computed tomography detection and automated quantification of systemic sclerosis-associated interstitial lung disease: preliminary study.

Lung ultrasound (LUS) is a promising tool for detecting systemic sclerosis-associated interstitial lung disease (SSc-ILD). Currently, consensus on the best LUS findings and execution technique is lacking. OBJECTIVES: To compare qualitative and quantitative assessment of B-lines and pleural line (PL) alterations in SSc-ILD with chest computed tomography (CT) analysis. METHODS: During 2021-2022, consecutive SSc patients according to 2013 ACR/EULAR classification criteria underwent pulmonary functional tests (PFTs). On the same day, if a CT was performed over a ± 6 months period, LUS was performed by two certified blinded operators using a 14-scans method. The ≥10 B-lines cut-off proposed by Tardella and the Fairchild's PL criteria fulfilment were selected as qualitative findings. As quantitative assessment, total B-lines number and the quantitative PL score adapted from the semi-quantitative Pinal-Fernandez score were collected. CT scans were evaluated by two thoracic radiologists for ILD presence, with further processing by automated texture analysis software (qCT). RESULTS: 29 SSc patients were enrolled. Both qualitative LUS scores were significantly associated to ILD presence on CT, with Fairchild's PL criteria resulting in slightly more accuracy. Results were confirmed on multivariate analysis. All qualitative and quantitative LUS findings were found to be significantly associated with qCT ILD extension and radiological abnormalities. Mid and basal PL quantitative score correlated with mid and basal qCT ILD extents. Both B-lines and PL alterations differently correlated with PFTs and clinical variables. CONCLUSION: This preliminary study suggests the utility of a comprehensive LUS assessment for SSc-ILD detection compared with CT and qCT.

Clinical Efficacy of Carbocysteine in COPD: Beyond the Mucolytic Action.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with a versatile and complicated profile, being the fourth most common single cause of death worldwide. Several research groups have been trying to identify possible therapeutic approaches to treat COPD, such as the use of mucoactive drugs, which include carbocysteine. However, their role in the treatment of patients suffering from COPD remains controversial due to COPD's multifaceted profile. In the present review, 72 articles, published in peer-reviewed journals with high impact factors, are analyzed in order to provide significant insight and increase the knowledge about COPD considering the important contribution of carbocysteine in reducing exacerbations via multiple mechanisms. Carbocysteine is in fact able to modulate mucins and ciliary functions, and to counteract viral and bacterial infections as well as oxidative stress, offering cytoprotective effects. Furthermore, carbocysteine improves steroid responsiveness and exerts anti-inflammatory activity. This analysis demonstrates that the use of carbocysteine in COPD patients represents a well-tolerated treatment with a favorable safety profile, and might contribute to a better quality of life for patients suffering from this serious illness.

A life-threatening small bowel obstruction as onset of an unknown sarcoidosis: A case report.

Sarcoidosis is a systemic granulomatous disorder of unknown etiology characterized by non-caseating granulomas at the site of disease. A confident diagnosis should be established by the evidence of typical granulomas on biopsy and after exclusion of other conditions. Clinically recognizable Gastrointestinal involvement (GI) occurs in less than 1.6% of patients with sarcoidosis, with data revealing small intestine participation in 0.03% of the cases and few anecdotal reports describe a peritoneal presentation. Clinical manifestations of peritoneal sarcoidosis are abdominal discomfort, bloating, weight loss, epigastric and peri-umbilical pain with or without ascites, bowel obstruction. Treatment depends on symptoms and disease activity. Herein we describe the case of a 42-years-old male patient who developed an acute, life-threatening small bowel obstruction as first manifestation of sarcoidosis. To the best of our knowledge, this is the only report showing such extensive and acute onset of intra-abdominal sarcoidosis in the absence of a previous disease manifestation and without pulmonary involvement.

Reduced ventilatory efficiency during exercise predicts major vascular complications and mortality for interstitial lung disease in systemic sclerosis.

OBJECTIVES: Major vascular complication, such as digital ulcers (DUs), pulmonary arterial hypertension (PAH) and scleroderma renal crisis (SRC) are hallmarks of systemic sclerosis (SSc). Interstitial lung disease (ILD) is the major cause of mortality in SSc. The aim of study is to identify cardiopulmonary exercise testing (CPET) variables that predict MVC and mortality for ILD in SSc patients. METHODS: In this cohort study, 45 SSc patients underwent clinical evaluation, echocardiography, pulmonary function tests (PFTs), high resolution computerised tomography (HRCT) and CPET. PFTs and echocardiography were performed annually for a 5-year follow-up. RESULTS: 16 (35.6%) SSc patients had MVC: 14 new DUs (31.1%), 1 PAH (2.2%) and 1 SRC (2.2%). At univariate regression analysis, mRss [HR 1.099 (1.008-1.199), p<0.05], NVC patterns (active and late) [HR 0.032 (0.004-0.250), p<0.001], V'E/V'CO2 slope [HR 1.123 (1.052-1.198), p<0.001] were predictive of new onset of MVC. In multivariate analysis, NVC patterns (active and late) (HR 0.044 (0.004-0.486), p<0.05), V'E/V'CO2 (HR 1.094 (1.020-1.198), p<0.05) were predictive of new onset of MVC. The 5-year mortality for ILD is 8.9%. In univariate analysis, DLco [(HR 0.927(CI 0.874- 0.983), p<0.05], V'E/V'CO2 slope and lung parenchymal with radiological patterns of ILD [(1.2.02 (CI 1.018-1.419), p<0.05], represent risk factors for 5-year mortality for ILD [HR 1.142 (1.030-1.267), p<0.05]. In multivariate analysis, only V'E/V'CO2 slope [1.268 (CI 1.003-1.602), p<0.05] represents a risk factor for 5-year mortality for ILD. CONCLUSIONS: V' E/V' CO2 slope is a prognostic marker of MVC and five-year mortality for ILD.

Mycophenolate Mofetil Improves Exercise Tolerance in Systemic Sclerosis Patients with Interstitial Lung Disease: A Pilot Study.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune disease characterized by the overproduction of collagen leading to fibrosis of the skin and internal organs. Interstitial lung disease (ILD) is one of the major causes of death in patients with SSc. Exercise tolerance can be investigated by cardio-pulmonary exercise testing (CPET). First-line therapies in patients with SSc associated with ILD (SSc-ILD) include cyclophosphamide and mycophenolate mofetil (MMF). The aim of this study was to evaluate the response of patients with SSc-ILD to MMF by means of CPET. METHODS: Ten consecutive SSc patients were enrolled in this study. All SSc patients underwent clinical evaluation, echocardiography, pulmonary function tests, high-resolution computed tomography (HRCT) and CPET at baseline and after 2 years of therapy with MMF. RESULTS: After 24 months of treatment with MMF (target dose 1500 mg twice daily), forced vitality capacity, diffusing capacity of the lungs for carbon monoxide and systolic pulmonary arterial pressure had not improved significantly and there were no significant differences in HRCT findigns. In addition, peak oxygen uptake (V'O2 peak) and ventilatory equivalents for carbon dioxide production (V'E/V'CO2 slope) had not improved significantly. In contrast, there was a significant improvement from baseline to 24 months of treatment in the respiratory exchange ratio [median (interquartile range): 1.07 (0.92-1.22) vs. 1.26 (1.22-1.28), respectively; p < 0.01] and in the Borg scale for leg discomfort [median (interquartile range): 5 (5-7) vs. 4 (3-4), respectively; p < 0.01] . CONCLUSION: These data from our pilot study on a small cohort of SSc patients are the first to demonstrate that treatment with MMF can improves exercise tolerance and leg discomfort in patients with SSc-ILD. These preliminary results need to be confirmed in large randomized studies.

Assessment of interstitial lung disease in systemic sclerosis using the quantitative CT algorithm CALIPER.

Interstitial lung disease (ILD) remains a major cause of morbidity and mortality in systemic sclerosis (SSc). Study aim is to characterize and quantify SSc-ILD by using Computer-Aided Lung Informatics for Pathology Evaluation and Rating (CALIPER). Secondly, our objective is to evaluate which radiological pattern is predictive of lung function decline at 12 months follow-up. In the prospective study (IRB 5435), 66 SSc patients underwent high-resolution computerized tomography (HRCT) at baseline. HRCT was performed according to standard protocol using a CT 64GE light speed VCT power scanner. CALIPER classified lung parenchyma on volume units. Every volume unit was classified into radiological parenchymal patterns (honeycombing, reticular and ground glass). Pulmonary function tests (PFTs) were performed at baseline and after 12 months of follow-up. Cigarette smoking and other lung diseases unrelated to SSc are exclusion criteria. CALIPER analysis showed normal lung parenchyma 87.4 ± 9.8%, ground glass 2.8 ± 5.3%, reticular 4 ± 5.7%, and honeycombing 1 ± 1%. In multiple regression analysis, FEV1 (p < 0.0001), FVC (p = 0.001), and DLCO (p < 0.0001) measurements at baseline showed a negative correlation with the reticular pattern percentage. At follow-up, DLCO reduction showed a positive correlation (p < 0.001) with the percentage of ground glass pattern (r = 0.33, beta coefficient = 0.51). In the ROC curve analysis, ground glass score is a good predictor (0.75, p = 0.009; 95% CI 0.59-0.91) of DLCO worsening, defined as a decrease of more than 10% of DLCO. Using a cutoff ≥ 4.5 for ground glass score, the RR for DLCO worsening is 6.8 (p < 0.01; 95% CI 1.6-29.2). The results of this study show that CALIPER is useful not only for quantifying lung damage but also for assessing worsening PFTs, but larger studies are needed to confirm these preliminary data.Key Points• At baseline reticular pattern showed negative correlation with PFTs• At follow-up ground glass pattern predicts worsening of DLCO• CALIPER is a useful to quantify lung damage.

Rational design, preparation and characterization of recombinant Ag85B variants and their glycoconjugates with T-cell antigenic activity against Mycobacterium tuberculosis.

Tuberculosis is the deadliest infectious disease in the world. The variable efficacy of the current treatments highlights the need for more effective agents against this disease. In the past few years, we focused on the investigation of antigenic glycoconjugates starting from recombinant Ag85B (rAg85B), a potent protein antigen from Mycobacterium tuberculosis. In this paper, structural modifications were rationally designed in order to obtain a rAg85B variant protein able to maintain its immunogenicity after glycosylation. Lysine residues involved in the main T-epitope sequences (namely, K30 and K282) have been substituted with arginine to prevent their glycosylation by a lysine-specific reactive linker. The effectiveness of the mutation strategy and the detailed structure of resulting neo-glycoconjugates have been studied by intact mass spectrometry, followed by peptide and glycopeptide mapping. The effect of K30R and K282R mutations on the T-cell activity of rAg85B has also been investigated with a preliminary immunological evaluation performed by enzyme-linked immunospotting on the different variant proteins and their glycosylation products. After glycosylation, the two variant proteins with an arginine in position 30 completely retain the original T-cell activity, thus representing adequate antigenic carriers for the development of efficient glycoconjugate vaccines against tuberculosis.

Alpha-1 Antitrypsin Deficiency: Current Perspective from Genetics to Diagnosis and Therapeutic Approaches.

Alpha-1 antitrypsin (A1AT) is a 52-kDa, acute phase glycoprotein encoded by the protease inhibitor (PI) locus, located on the long arm of chromosome 14 (14q31-32.3). Its structure is composed of a total of 7 exons, 4 coding (II, III, IV, and V) and 3 non-coding (IA, IB, and IC). A1AT is produced primarily by hepatocytes and acts as a serine protease inhibitor with antiprotease and immunoregulatory activities. The main target of A1AT is neutrophil elastase (NE), an enzyme released during a neutrophil-mediated inflammatory process. When the enzyme is not adequately balanced by A1AT activity, it can cause tissue injury and destruction. A1AT deficiency (A1ATD) is a genetic autosomal recessive disease, characterized by low serum levels of A1AT. The condition may lead to liver disease, early-onset pulmonary emphysema and, rare multi-organ vasculitis, necrotizing panniculitis and fibromyalgia. At least 100 allelic variants of the polymorphic PI locus have been described with groups including associations with different A1AT plasma levels and functions. Treatments with purified A1AT preparations, obtained through pooled human plasma (augmentation therapy), have been proven to improve survival and disease-related quality of life, as well as, slow down the progression of organ damage. Furthermore, ongoing research is now focusing on the development of specifically targeted, new medications. The aim of this review is to summarize our knowledge of the genetic A1AT variants, focusing on their variable clinical manifestation, report routine and recently updated laboratory diagnostic techniques, and to highlight the relevance of early diagnosis of A1ATD. Moreover, we will review the role of augmentation therapy recommendations and future perspectives focusing on a personalized treatment of A1ATD.

An unusual outbreak of nontuberculous mycobacteria in hospital respiratory wards: Association with nontuberculous mycobacterial colonization of hospital water supply network.

The incidence and prevalence of pulmonary nontuberculous mycobacterial (NTM) infection is increasing worldwide arousing concerns that NTM infection may become a serious health challenge. We recently observed a significant increase of NTM-positive sputa samples from patients referred to respiratory disease wards of a large tertiary hospital in Rome. A survey to identify possible NTM contamination revealed a massive presence of NTM in the hospital water supply network. After decontamination procedures, NTM presence dropped both in water pipelines and sputa samples. We believe that this observation should encourage water network surveys for NTM contamination and prompt decontamination procedures should be considered to reduce this potential source of infection.

Molecular characterization of a selected cohort of patients affected by pulmonary metastases of malignant melanoma: Hints from BRAF, NRAS and EGFR evaluation.

BACKGROUND: Melanoma is highly curable in early stages but holds devastating consequences in advanced phases with a median survival of 6-10 months. Lungs are a common metastasis target, but despite this, limited data are available on the molecular status of pulmonary lesions. MATERIALS AND METHODS: 25 patients with surgically resected melanoma lung metastases were screened for BRAF, NRAS, CKIT and EGFR alterations. The results were correlated with time to lung metastasis (TLM), relapse-free survival after metastasectomy (RFS) and overall survival (OS). RESULTS: BRAF or NRAS were mutated in 52% and 20% of cases while CKIT was unaffected. Chromosome 7 polysomy was detected in 47% of cases with 17.5% showing EGFR amplification and concomitant BRAF mutation. NRAS mutated patients developed LM within 5 yrs from primary melanoma with larger lesions compared with BRAF (mean diameter 3.3 ± 2.2cm vs 1.9 ± 1.1cm, p = 0.2). NRAS was also associated with a shorter median RFS and OS after metastasectomy. Moreover, Cox regression analysis revealed that NRAS mutation was the only predictive factor of shorter survival from primary melanoma (p = 0.039, OR = 5.5 (1.1-27.6)). CONCLUSIONS: Molecular characterization identifies advanced melanoma subgroups with distinct prognosis and therapeutic options. The presence of NRAS mutation was associated to a worse disease evolution.

[Rare lung diseases]. / Le malattie polmonari rare.

"Rare" diseases are a family of different disorders or conditions affecting fewer than 1/2000 in the general population. Among them, 150 involve respiratory system. To date, most reviews in literature focuses on the more important pulmonary lung diseases without taking in account the overall framework and diagnostic and therapeutic algorithms that allow a correct comprehensive approach to these disorders. The aim of our review is to describe a possible global approach to the management of these diseases trying to take a picture of the overall complexity of the field. We also want to highlight the role played by research on rare diseases pathogenesis in revealing, at least in part, mechanisms underlying many of "more common" disorders with the purpose to update the current state of knowledge.

Long-term home noninvasive mechanical ventilation increases systemic inflammatory response in chronic obstructive pulmonary disease: a prospective observational study.

BACKGROUND: Long-term home noninvasive mechanical ventilation (NIV) is beneficial in COPD but its impact on inflammation is unknown. We assessed the hypothesis that NIV modulates systemic and pulmonary inflammatory biomarkers in stable COPD. METHODS: Among 610 patients referred for NIV, we shortlisted those undergoing NIV versus oxygen therapy alone, excluding subjects with comorbidities or non-COPD conditions. Sputum and blood samples were collected after 3 months of clinical stability and analyzed for levels of human neutrophil peptides (HNP), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha). Patients underwent a two-year follow-up. Unadjusted, propensity-matched, and pH-stratified analyses were performed. RESULTS: Ninety-three patients were included (48 NIV, 45 oxygen), with analogous baseline features. Sputum analysis showed similar HNP, IL-6, IL-10, and TNF-alpha levels (P > 0.5). Conversely, NIV group exhibited higher HNP and IL-6 systemic levels (P < 0.001) and lower IL-10 concentrations (P < 0.001). Subjects undergoing NIV had a significant reduction of rehospitalizations during follow-up compared to oxygen group (P = 0.005). These findings were confirmed after propensity matching and pH stratification. CONCLUSIONS: These findings challenge prior paradigms based on the assumption that pulmonary inflammation is per se detrimental. NIV beneficial impact on lung mechanics may overcome the potential unfavorable effects of an increased inflammatory state.

Demographic characteristics, nicotine dependence, and motivation to quit as possible determinants of smoking behaviors and acceptability of shocking warnings in Italy.

INTRODUCTION: This paper presents the final results of a cross-sectional study started in 2010. It compares the perceived efficacy of different types of tobacco health warning (texts versus shocking pictures) to quit or reduce tobacco use. METHODS: The study conducted between 2010 and 2012 in Italy enrolled adults smokers. Administering a questionnaire demographic data, smokers behaviors were collected. Showing text and graphic warnings (the corpse of a smoker, diseased lungs, etc.) the most perceived efficacy to reduce tobacco consumption or to encourage was quit. RESULTS: 666 subjects were interviewed; 6% of responders referred that they stopped smoking at least one month due to the textual warnings. The 81% of the smokers perceived that the warnings with shocking pictures are more effective in reducing/quitting tobacco consumption than text-only warnings. The younger group (<45 years), who are more motivated to quit (Mondor's score ≥ 12), and females showed a higher effectiveness of shocking warnings to reduce tobacco consumption of, 76%, 78%, and 43%, respectively with P < 0.05. CONCLUSIONS: This study suggests that pictorial warnings on cigarette packages are more likely to be noticed and rated as effective by Italian smokers. Female and younger smokers appear to be more involved by shock images. The jarring warnings also appear to be supporting those who want to quit smoking. This type of supportive information in Italy may become increasingly important for helping smokers to change their behavior.

Implementation of smoking ban: a survey in a public hospital setting.

Smoking in hospitals is banned in most of European countries; nevertheless, implementing a total smoking ban is particularly difficult and policy breaches are frequent. Aim of our study was to monitor the compliance with the smoke-free policy within a hospital district by measuring particulate matters (PM2.5). We designed an observational study and identified six sensitive locations within the hospitals: surgical units, administrative offices, hall, outdoor main entrances and as controls an outdoor and an indoor area. To rule out potential confounders we included in the evaluation the roadways surrounding the hospital district. PM2.5 median concentrations observed in outdoor main entrances and in hall were significantly higher (16.4 and 13.4 µg m(-3)), as compared with the other settings (P < 0.0001). This data warrant an implementation of current policies to protect patients, visitors and employees from passive second-hand smoke leading to a smoking prohibition in any hospital surroundings.

A 6MWT index to predict O2 flow correcting exercise induced SpO2 desaturation in ILD.

INTRODUCTION: Ambulatory oxygen (O2) is prescribed to interstitial lung disease (ILD) patients with mild hypoxemia, breathlessness and dyspnea on exertion. Oxygen titration is generally done with the 6 minute walk test (6MWT) to determine the O2 flow preventing oxygen saturation by pulse oximetry (SpO2) from falling below 88%. His study was designed to generate a 6MWT index predicting the O2 flow allowing completion of the 6MWT without oxygen desaturation. METHODS: Oxygen titration data from a group of 66 ILD patients and 30 controls, were used to generate the algorithm determining an index (O2-GAP) predicting oxygen flow required to complete a 6MWT without desaturation below 88%. This index was validated in a group of 93 ILD patients. RESULTS: The O2-GAP index, as obtained from the derivation population, (r(2) = 0.97, p < 0.001) was shown to correctly predict the oxygen flow required to complete the 6MWT without SpO2 falling below 88% validated in the validation population (r(2) = 0.842; p < 0.001). CONCLUSIONS: The O2-GAP index appears to be a useful tool to titrate ambulatory O2 with a single 6MWT on room air in ILD patients with breathlessness and dyspnea on exertion.

Use of discriminant analysis in assessing pulmonary function worsening in patients with sarcoidosis by a panel of inflammatory biomarkers.

OBJECTIVES AND DESIGN: To date, no sufficiently sensitive and specific single marker has been found to predict the clinical course of sarcoidosis. We designed a cohort study to investigate whether a panel of biomarkers measured in bronchoalveolar lavage (BAL) and peripheral blood could help predict pulmonary function worsening during the clinical course of sarcoidosis. METHODS: We analyzed 30 individuals with histologically proven sarcoidosis. At baseline, participants underwent pulmonary function tests (PFTs), fiberoptic bronchoscopy and radiological investigations. BAL and blood cellular profiles were obtained from all individuals and six pro-inflammatory molecules were quantified in BAL and serum. PFTs were performed at follow-up visits over a 2-year period. Using discriminant function analysis, a canonical variable was generated to optimize the accuracy of selected variables in predicting pulmonary function worsening and was validated on a subset of nine consecutive individuals with sarcoidosis. RESULTS: A combination of 6 markers from BAL was able to predict pulmonary function worsening in 96 % of patients [95 % confidence interval (CI) 84.4-99.81]. We validated the generated formula on a group of nine patients with sarcoidosis, obtaining 77.8 % correct classification (95 % CI 45.3-93.7). CONCLUSIONS: Our results show that a combinational approach could contribute to identifying individuals likely to experience pulmonary function worsening, thus helping to decide the correct therapeutic strategies.

Mixed acid-base disorders, hydroelectrolyte imbalance and lactate production in hypercapnic respiratory failure: the role of noninvasive ventilation.

BACKGROUND: Hypercapnic Chronic Obstructive Pulmonary Disease (COPD) exacerbation in patients with comorbidities and multidrug therapy is complicated by mixed acid-base, hydro-electrolyte and lactate disorders. Aim of this study was to determine the relationships of these disorders with the requirement for and duration of noninvasive ventilation (NIV) when treating hypercapnic respiratory failure. METHODS: Sixty-seven consecutive patients who were hospitalized for hypercapnic COPD exacerbation had their clinical condition, respiratory function, blood chemistry, arterial blood gases, blood lactate and volemic state assessed. Heart and respiratory rates, pH, PaO(2) and PaCO(2) and blood lactate were checked at the 1st, 2nd, 6th and 24th hours after starting NIV. RESULTS: Nine patients were transferred to the intensive care unit. NIV was performed in 11/17 (64.7%) mixed respiratory acidosis-metabolic alkalosis, 10/36 (27.8%) respiratory acidosis and 3/5 (60%) mixed respiratory-metabolic acidosis patients (p = 0.026), with durations of 45.1 ± 9.8, 36.2 ± 8.9 and 53.3 ± 4.1 hours, respectively (p = 0.016). The duration of ventilation was associated with higher blood lactate (p<0.001), lower pH (p = 0.016), lower serum sodium (p = 0.014) and lower chloride (p = 0.038). Hyponatremia without hypervolemic hypochloremia occurred in 11 respiratory acidosis patients. Hypovolemic hyponatremia with hypochloremia and hypokalemia occurred in 10 mixed respiratory acidosis-metabolic alkalosis patients, and euvolemic hypochloremia occurred in the other 7 patients with this mixed acid-base disorder. CONCLUSIONS: Mixed acid-base and lactate disorders during hypercapnic COPD exacerbations predict the need for and longer duration of NIV. The combination of mixed acid-base disorders and hydro-electrolyte disturbances should be further investigated.

Long-term disease-free survival of a patient with synchronous bilateral lung adenocarcinoma displaying different EGFR and C-MYC molecular characteristics.

The main difficulty with multiple lung tumors is distinguishing synchronous and metachronous lesions from second independent primary tumors, particularly when dealing with the same histologic type. Challenging diagnostic hurdles may explain, at least in part, the extremely variable (0%-79%) 5-year survival rate. We present a case report of a patient with synchronous primary adenocarcinoma treated with surgery that exhibited different EGFR gene status, with an exon 19 mutation in the adenocarcinoma of the left upper lobe that was absent in the right upper lobe. Further, specific EGFR and C-MYC amplification events were associated only with the EGFR-mutated lesion. According to an independent evolution theory, these events were classified as early stage, and the patient is still alive and free of disease 70 months after surgery. Molecular evaluation was an important tool to support the diagnosis of synchronous primary adenocarcinoma, which had not been possible with the application of Martini-Melamed criteria.