Identification of oestrogen, progesterone receptor and human epidermal growth factor receptor 2 expression in mediastinal metastases of breast cancer obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

BACKGROUND: In breast cancer patients, the expression statuses of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are crucial in the choice of treatment. Receptor expression in metastatic lesions can differ from the primary tumour. The aim of our study was to analyse the utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain samples allowing the identification of ER, PR and HER2 expression in patients with mediastinal metastases of breast cancer. PATIENTS AND METHODS: The clinical files of all patients with a final diagnosis of breast cancer mediastinal metastases diagnosed by EBUS-TBNA in our institution were retrospectively analysed. The ability of EBUS-TBNA to obtain samples that allowed hormone receptor and HER2 expression analysis was calculated. RESULTS: Twenty-four patients were included. ER, PR and HER2 assessments could be performed in 22, 20 and 22 patients, respectively. In 20 of the 24 patients it was possible to investigate all three types of receptor expression. In the remaining four cases, where ER, PR or HER2 expression tests could not be performed, it was due to a lack of tissue. In cases with adequate results for EBUS-TBNA and the primary tumour agreement was greater for ER (16/19) and HER2 (12/14) than PR (8/17). Based on receptor status, there was a change in the choice of treatment for five patients. CONCLUSION: In patients with breast cancer mediastinal metastases, ER, PR and HER2 expression can be assessed in samples obtained by EBUS-TBNA whenever a sufficient tissue sample is collected.

Condylomata, cytological abnormalities and human papillomavirus infection in the anal canal in HIV-infected men.

BACKGROUND: Genital infections with low-risk (LR) and high-risk (HR) human papillomavirus (HPV) genotypes are associated with ano-genital condylomata and anal squamous cell cancer. HPV-related pathologies in HIV-infected men are a serious concern. In this study, the prevalence of anal condylomata and their association with cytological abnormalities and HPV infection in the anal canal in HIV-infected men [men who have sex with men (MSM) and heterosexuals] were estimated. METHODS: This was a cross-sectional study based on the first visits of patients in the Can Ruti HIV-positive Men (CARH·MEN) cohort. Anal condylomata were assessed by clinical and proctological examination. Samples from the anal canal were collected for HPV genotyping and cytological diagnoses. RESULTS: A total of 640 HIV-infected men (473 MSM and 167 heterosexuals) were included in the study. The overall prevalence of anal condylomata was 25% [157 of 640; 95% confidence interval (CI) 21-28%]; in MSM it was 28% and in heterosexuals it was 15% [odds ratio (OR) 2.2; 95% CI 1.4-3.5]. In patients with anal condylomata, HPV infection in the anal canal was more prevalent (92% vs. 67% in those without anal condylomata; OR 8.5; 95% CI 3.2-22). This higher HPV prevalence involved at least two HPV genotypes (OR 4.0; 95% CI 2.2-7.1), mainly HR genotypes (OR 3.3; 95% CI 1.7-6.4). Similarly, the cumulative prevalence of HPV-6 and HPV-11 was higher in patients with anal condylomata (63% vs. 19% in those without anal condylomata). Having anal condylomata was associated with higher prevalences of cytological abnormalities (83% vs. 32% in those without anal condylomata; OR 6.9; 95% CI 3.8-12.7) and high-grade squamous intraepithelial lesions (HSILs) (9% vs. 3% in those without anal condylomata; OR 9.0; 95% CI 2.9-28.4) in the anal canal. CONCLUSIONS: HIV-infected men with anal condylomata were at risk of presenting HSILs and harbouring multiple HR HPV infections in the anal canal. Although MSM presented the highest prevalence of anal condylomata, heterosexual men also had a clinically important prevalence. Our findings emphasize the importance of screening and follow-up for condylomata in the anal canal in HIV-infected men.

Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations.

The presence of somatic mutations of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in patients with advanced nonsmall cell lung cancer (NSCLC) correlates with a good response to tyrosine kinase inhibitors. The usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the detection of EGFR mutations in cells recovered from malignant mediastinal nodes in patients with NSCLC was assessed. All patients with lung adenocarcinoma or unspecified NSCLC referred for staging with EBUS-TBNA were included. Nodes with a short-axis diameter of >5 mm were sampled, and genomic DNA from metastatic tumour cells was obtained for analysis of exons 19 and 21. The impact of sampling on management was assessed. EGFR gene analysis of the EBUS-TBNA sample was feasible in 26 (72.2%) out of the 36 patients with lymph node metastasis. Somatic mutations of the EGFR gene were detected in tissue obtained through EBUS-TBNA in two (10%) out of 20 patients with metastasic lung adenocarcinoma. Malignant tissue samples obtained by EBUS-TBNA from patients with nodal metastasis of NSCLC are suitable for the detection of EGFR mutations in most cases, and this technique demonstrates mutated neoplastic cells in a tenth of patients with adenocarcinoma.

Aberrant gene methylation and bronchial dysplasia in high risk lung cancer patients.

INTRODUCTION: The risk for lung cancer is incremented in high degree dysplasia (HGD) and in subjects with hypermethylation of multiple genes. We sought to establish the association between them, as well as to analyze the DNA aberrant methylation in sputum and in bronchial washings (BW). METHODS: Cross sectional study of high risk patients for lung cancer in whom induced sputum and autofluorescence bronchoscopy were performed. The molecular analysis was determined on DAPK1, RASSF1A and p16 genes using Methylation-specific PCR. RESULTS: A total of 128 patients were enrolled in the study. Dysplasia lesions were found in 79 patients (61.7%) and high grade dysplasia in 20 (15.6%). Ninety eight patients out of 128 underwent molecular analysis. Methylation was observed in bronchial secretions (sputum or BW) in 60 patients (61.2%), 51 of them (52%) for DAPK1, in 20 (20.4%) for p16 and in three (3.1%) for RASSF1A. Methylated genes only found in sputum accounted for 38.3% and only in BW in 41.7%, and in both 20.0%. In the 11.2% of the patients studied, HGD and a hypermethylated gene were present, while for the 55.1% of the sample only one of both was detected and for the rest of the subjects (33.6%), none of the risk factors were observed. CONCLUSIONS: Our data determines DNA aberrant methylation panel in bronchial secretions is present in a 61.2% and HGD is found in 15.6%. Although both parameters have previously been identified as risk factors for lung cancer, the current study does not find a significative association between them. The study also highlights the importance of BW as a complementary sample to induced sputum when analyzing gene aberrant methylation.

A distinctive cytologic pattern for diagnosing tuberculous lymphadenitis in AIDS.

Tuberculous lymphadenitis (TL) is a very common infection in human immunodeficiency virus (HIV)-infected patients. We performed fine-needle aspiration biopsy (FNAB) of enlarged lymph nodes in 57 HIV-infected patients to evaluate its usefulness in this population. We observed three cytologic patterns in 21 patients diagnosed as having TL: granulomatous lymphadenitis (GL) in 4 FNABs, necrotizing granulomatous lymphadenitis (NGL) in 7 FNABs, and necrotizing lymphadenitis (NL) in 12 FNABs. GL and NGL are already well-known and considered to be highly suggestive of TL. Our results support the idea that NL should have the same diagnostic value as GL or NGL. In the group of 12 patients with NL, TL was confirmed in 11 by microbiologic methods (7 by a positive Ziehl-Neelsen stain and 4 by a positive Löwenstein culture) and in the remaining patient by a biopsy that showed NGL with acid-fast bacilli. We conclude that FNAB is a useful, inexpensive, and safe technique for diagnosing TL in HIV-infected patients. The finding of a NL pattern is suggestive enough of TL to start antituberculous treatment.

Fine-needle aspiration cytology of benign nodular thyroid disease. Value of re-aspiration.

Fine-needle aspiration cytology (FNAC) has become a widespread procedure for the study of thyroid nodules (TN). Some authors recommend the practice of repeated punctures for their follow-up. This study was done to determine the usefulness of repeated FNAC in patients with benign nodular thyroid disease. We have studied 251 fine-needle re-aspirations performed on 116 females aged 45.6 +/- 14 years with benign nodular thyroid disease. The time elapsed between each consecutive FNAC was 1 year. No patients presented any changes in the size or consistency of their nodular goiters during this period; all FNACs were carried out by the same physician in the same thyroid area according to the Löwhagen technique, with a minimum of two or three aspirations of each nodule, and processed in the same way and valued by the same cytologist without any knowledge of previous cytological diagnoses. These were done using strictly classical criteria (Löwhagen). One hundred and five out of 116 patients (90.51%) with two consecutive FNACs (210) showed identical cytological diagnoses in the two specimens studied. The remaining 11 patients (9.48%) with two FNACs were diagnosed with colloid goiter and cyst alternately. Fifteen out of 19 patients (78.94%) with three FNACs showed identical cytological diagnoses in the three samples and the rest (21%) also demonstrated alternate diagnoses of colloid goiter and cyst. Our results show that the routine performance of repeated FNAC in the follow-up of females with benign nodular thyroid disease, without any clinical changes, is of limited usefulness.

Proliferating cell nuclear antigen expression in normal, regenerative, and neoplastic liver: a fine-needle aspiration cytology and biopsy study.

Information about a tissue's proliferative activity can be obtained from the immunocytochemical investigation of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta expressed by cycling cells. To determine whether a relationship exists between morphology and PCNA expression in normal, regenerative, and malignant neoplastic hepatocytes, this study was undertaken on 48 fine-needle aspiration cytology (FNAC) cell blocks from eight normal livers, eight cirrhotic livers, and 32 hepatocellular carcinomas (HCCs), as well as on 41 needle or wedge biopsy specimens from 10 normal livers, 13 cirrhotic livers, one focal nodular hyperplastic liver, and 17 HCCs. Anti-PCNA monoclonal antibody PC10 was applied to formalin-fixed, paraffin-embedded tissue using the avidin-biotin method. Proliferating cell nuclear antigen immunoreactivity was evaluated as follows: absent; minimal, less than 5% positive nuclei; grade 1, 5% to 25% positive nuclei; grade 2, 26% to 50% positive nuclei; grade 3, 51% to 75% positive nuclei; and grade 4, 76% to 100% positive nuclei. In both the FNAC and biopsy series normal and regenerative livers were either completely negative or minimally immunoreactive (under 5% positive nuclei). In contrast, all well-differentiated HCC cases exhibited over 15% positive nuclei. Most well-differentiated HCCs were grade 1 (85.7% in the FNAC series and 76.92% in the biopsy series) and the majority of moderately differentiated HCCs were grade 3 (63.63% in the FNAC series, but only 50% in the biopsy series). Therefore, absent or minimal PCNA immunoreactivity seems to be a useful adjuvant to discriminate normal/regenerative liver from HCC, whose degree of differentiation tends to correlate with the level of PCNA expression. These observations apply to both the FNAC and biopsy series, which yielded very similar data.

Kinetics of cell replication in the uterine cervix. VIII. Quantitation of chronologic events in proliferating loci.

The frequency and the topographic distribution of DNA replicating cervical basal squamous cells (as deduced by 3H-thymidine in vivo administration up to 8 hours before sacrifice) was studied in 70,000 basal cells in 70 C57B1 mice. While the number of labelled basal cells/animal was similar in those injected only once, a significantly higher number of labelled basal cells was recorded in animals injected twice or three times. Following one single injection, 30% (8 h previously) and 31% (1 h previously) of the DNA replicating basal cells occurred in pairs or in loci of three, four or more labelled cells. After repeated injections, the rates were 49% for those animals injected twice before sacrifice and 47% for those injected three times. Thus repeated injections resulted in a significantly increased number of loci of three, four or more DNA replicating basal cells when compared to those injected only once. The present results suggest that there is a chronological asynchrony in the DNA replication of proliferating basal cells-loci of the uterine cervix.

Fine-needle aspiration biopsy of metaplastic carcinoma of the breast: report of a case with abundant myxoid ground substance.

Metaplastic carcinoma (MC) is an uncommon neoplasm of the breast. There are several variants of MC depending on the dominant histologic pattern. The components include over infiltrating ductal carcinoma, extensive squamous differentiation and spindle cell proliferation with or without chondroid or asseous heterologous elements. In FNA smears, only 57% of cases show both ductal carcinoma and metaplastic component. Thus, in almost one half of the cases, the diagnosis is not possible by FNA. Often it is difficult to define the epithelial or sarcomatous character of malignant cells. We describe a case of metaplastic carcinoma of the breast studied by fine-needle aspiration cytology in which myxoid ground substance was the dominant feature in the cytology smears. The rest of the material was composed of scanty isolated atypical cells with large and irregular nuclei. It is important to bear in mind the diagnosis of MC and make a careful search for atypical cells when the cytological smears are mainly composed of myxoid ground substance.

Kinetics of cell replication of the uterine cervix. IV. Proliferative loci in the basal cell layer.

The sequence of dividing and nondividing cells along the basal cell layer of the uterine cervix of C57B1 mice was investigated eight hours after vincristine injection. Of the basal cells in mitosis, 42.8% were solitary and 57.2% in groups of two or more mitoses. In contrast, 99.2% of the mitoses occurring in untreated animals were solitary, and less than 1% were double. These results suggest the existence in the basal cell layer of loci of cell production composed of multiple mitoses. The multiple mitotic loci are not observed spontaneously, due to asynchrony in cell division.

Kinetics of cell replication in the uterine cervix. VII. Loci of mitotic basal cells during cervical carcinogenesis.

The frequency and the topographic occurrence (i.e., loci) of vincristine-arrested mitosis in the basal-cell layer was studied during the process of cervical carcinogenesis in C57B1 mice. The total frequency of mitosis decreased (by comparison to controls) not only in cervical intraepithelial neoplasias of grades I, II and III and in invasive carcinomas, but also in the normal epithelium of carcinogen-treated animals. This confirms earlier results and suggests that the pace of replication of cells in contact with the stroma is decreased during carcinogenesis. On the other hand, the proportion of the number of mitotic basal cells occurring singly or in groups of 2, 3 or greater than or equal to 4, as well as the proportion of loci with 1, 2, 3 or greater than or equal to 4 mitoses, were similar during cervical carcinogenesis (when compared to controls). It would thus appear that the proliferation of the cervical epithelium during carcinogenesis is regulated by two factors: one that seems to diminish the total number of mitotic basal cells during carcinogenesis, and another that seems to maintain as constant the proportion of mitotic loci, both under normal conditions and throughout the development of intraepithelial neoplasias, in the uterine cervix.

Kinetics of cell replication in the uterine cervix. VI. Loci of DNA-synthesizing cells and arrested mitoses in the basal-cell layer.

The mode of proliferation in the basal-cell layer of the squamous cervical epithelium was investigated in C57B1 mice with the aid of 3H-thymidine and vincristine. Six hours after vincristine injection and two hours after thymidine injection, 33% of the basal cells were in DNA synthesis and 12% in mitosis. Of these, only 23% of the cells in DNA synthesis and 45% of those in mitosis were found as single cells. The remaining cells proliferated in clusters of two or more cells. As many as 59% of the cells in DNA synthesis and 30% of those in mitosis occurred in colonies of three or more consecutive cells, indicating that multicell clustering is a rather common pattern of basal cell proliferation. Multicell loci of DNA-synthesizing cells occurred contemporaneously with but independently of multicell loci of mitotic cells (the loci were nonconsecutive). Basal-cell replication in the squamous cervical epithelium thus appears to be an organized process of cell renewal.

Kinetics of cell replication of the uterine cervix. I. Normal epithelium.

The rate of cell production in the squamous cervical epithelium, as deduced from the number of arrested mitoses at various time intervals after a single injection of vincristine, was investigated in 83 C57B1 mice. In the basal cell layer several peaks of arrested mitoses were registered: the first major peak was found at 9 hours, the second at 12 hours, the third at 16 hours and the fourth at 20 hours. Distinct peaks of arrested mitosis were also found in the nonbasal (parabasal) cell layers. Each mitotic peak may correspond to a number of cells that had entered the proliferative pool from the "resting" compartment. The stathmokinetic effect of vincristine on the squamous cervical epithelium had ceased after 20 hours.

p53 immunoreaction in hepatocellular carcinoma and its relationship to etiologic factors. A fine needle aspiration study.

OBJECTIVE: To determine immunohistochemically the expression of mutant p53 phosphoprotein in hepatocellular carcinoma (HCC) and its possible relationship to several etiologic factors. STUDY DESIGN: The study group consisted of 62 samples of HCC, grades 2, 3 and 4, obtained by fine needle aspiration cytology. The associated risk factors detected in these patients were as follows: ethanol abuse, ethanol abuse plus hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, HBV infection, HCV infection, non-A/ non-B hepatitis, hemochromatosis and obesity. RESULTS: Mutant p53 expression was identified in 22% of HCC and seemed to correlate with tumor grade. Positive immunostaining was frequently associated with a history of alcohol abuse (42%) and also with viral infection (HBV, 21%; HCV, 7%; non-A/non-B hepatitis, 7%). CONCLUSION: Mutant p53 seems to intervene in the progress of HCC through various grades of increasing malignancy. The association we found between alcohol intake and mutant p53 expression may deserve further investigation.

[Identifying asbestos bodies in bronchoalveolar lavage fluid]. / Identificación de cuerpos de asbesto en el lavado broncoalveolar.

UNLABELLED: Asbestos bodies (AB) in respiratory secretions in bronchoalveolar lavage (BAL) identify subjects with lower airway AB content is a potential cause of pleural or pulmonary disease. The precision of this qualitative measure, however, has not been adequately analyzed. OBJECTIVE: To determine the sensitivity and specificity of finding AB in BAL fluid by conventional qualitative cytology in comparison with the quantification of AB in BAL fluid. METHOD: BAL samples from 40 subjects exposed to asbestos (mean age 59.2 years; men/women 36/4) were processed in the following ways: 1) qualitative cytology and 2) quantification of AB in BAL fluid expressed as AB/ml. The concentration of AB in BAL fluid was considered the gold standard (upper limit of normal 1 AB/ml) for determining the precision of qualitative cytology. RESULTS: In 9 of the 40 cases (22.5%) AB was found in BAL liquid cytology, but in only five of them were AB counts greater than 1 AB/ml. AB counts also showed concentrations greater than 1 AB/ml for four patients whose qualitative results were negative. The sensitivity of a qualitative AB-positive finding for identifying subjects with potentially disease-causing AB concentrations was 0.55, while specificity was 0.87. We conclude that a qualitative finding of AB in BAL fluid is adequately specific, but that sensitivity is very low, an indication that AB concentration in BAL should be determined to adequately screen for patients at high risk of developing disease.

[Frequency and risk of bronchopulmonary neoplasia related to asbestos]. / Frecuencia y riesgo de neoplasia broncopulmonar relacionada con asbesto.

BACKGROUND: The goal of this study was to determine the prevalence of asbestos-related lung cancer and the importance of the occupational exposure to this inorganic fibre as a risk factor. PATIENTS AND METHODS: We performed a cross-sectional study of 82 patients with lung cancer (mean age 62 SD 9 years) and 53 patients without pleuropulmonary disease (63 SD 13 years). The occupational exposure to asbestos was determined by a questionnaire. We determined the concentration of asbestos bodies (AB) in bronchoalveolar lavage (BAL) (93 patients) or lung tissue (42 patients) after chemical digestion, with the results being expressed as AB/mL BAL or AB/g dry lung, respectively. A concentration higher than 1 AB/mL or 1,000 AB/g was considered as marker of high asbestos burden in lung tissue, which could be potentially responsible for pleuropulmonary disease. The importance of asbestos occupational exposure as a risk factor for lung cancer was determined using logistic regression models. RESULTS: 25 patients with lung cancer reported occupational exposure to asbestos (30%) and in 13 out of them AB were detected in BAL or lung tissue (24%), at high concentrations in 3 cases (4%). Six patients from the group without pleuropulmonary disease reported occupational exposure to asbestos (11%) and in 13 out of them AB were found in some samples (24%), with no case having high concentrations. In the univariate logistic regression analysis, diagnosis of bronchial neoplasia was associated with both smoking (OR 10.10, 95% CI 3.50-29.13) and occupational exposure to asbestos (OR 3.69, 95% CI 1.39-9.77). The association between asbestos exposure and lung cancer persisted statistically significant after adjustment for smoking (OR 2.80, 95% CI 1.00-7.84). CONCLUSION: In Spain, lung cancer was related to occupational exposure to asbestos in 4% of cases, and it appeared to exist a synergistic effect of smoking. Occupational exposure to this inorganic fibre doubles the risk of suffering from lung cancer.

Representativeness of nodal sampling with endobronchial ultrasonography in non-small-cell lung cancer staging.

The objective of our study was to determine the procedure-related requirements of mediastinal node sampling with endobronchial ultrasonography with real-time transbronchial needle aspiration (EBUS-TBNA) that would provide negative predictive value (NPV) for the identification of stage III disease in non-small-cell lung cancer (NSCLC) high enough to consider the technique equivalent to cervical mediastinoscopy. Representative EBUS-TBNA was defined as a sampling procedure obtaining satisfactory samples from normal nodes in regions 4R, 4L and 7 or diagnosing malignancy in mediastinal nodes. NPV was estimated using the results of postsurgical staging in patients who underwent surgery as a reference. Two-hundred ninety-six patients staged with EBUS-TBNA were included. Representative samples from regions 4R, 4L and 7 showing nonmalignant cytology were obtained from 98 patients (33.1%) and EBUS-TBNA detected N2/N3 disease in 150 (50.7%). Accordingly, an EBUS-TBNA procedure accomplishing the representativeness criteria required for sampling was attained in 248 of the participating patients (83.8%). The NPV of the procedure in this setting was 93.6%, with false-negative results only found in 5 patients, four of them with nodal metastasis out of the reach of EBUS-TBNA (regions 5, 8 and 9). In conclusion, representative sampling of regions 4R, 4L and 7 is achieved in more than 80% of patients staged using EBUS-TBNA, and in the procedures that attain this requirement a NPV >90% for mediastinal malignancy is reached, a figure equivalent to cervical mediastinoscopy.

Human papillomavirus HPV-16, 18, 52 and 58 integration in cervical cells of HIV-1-infected women.

BACKGROUND: Genomic integration of high-risk human papillomavirus into the cellular genome is considered an important event in the pathogenesis of cervical cancer related to the progression from premalignant cervical lesions to invasive cervical carcinoma. OBJECTIVE: This cross-sectional study was aimed to characterize the viral integration of HPV-16, HPV-18, HPV-52 and HPV-58 in cervical cells. STUDY DESIGN: HPV genotypes were determined by PCR and HPV integration by multiplex PCR in HIV-1-infected women without a background of HPV-related pathology. RESULTS: This study included 251 cervical cells samples of consecutive HIV-positive women who were visited between 1999 and 2003. The overall prevalence of HPV infection was 53% (133/251, 95%CI: 47-59%). The most prevalent genotypes were HPV-16 (27%), HPV-33 (15%), HPV-52 (8%) and HPV-58 (8%). The prevalence of abnormal cervical cytology was 33% (83/251, 95%CI: 27-39%). The overall prevalence of HPV integration was 11% (27/251, 95%CI: 7-15%), and the prevalence of HPV-16 integration was 33% (22/67, 95%CI: 22-45%), HPV-18 integration was 30% (3/10, 95%CI: 7-65%) and HPV-52 integration was 10% (2/19, 95%CI: 1-32%). No HPV-58 integration was detected. The percentage of HPV-16 and HPV-18 integration increased with the severity of the cervical lesions, HPV-16 integration was almost 70% and HPV-18 integration was 50% in high-grade squamous intraepithelial lesions. Integration was the most important risk factor associated with cervical dysplasia (OR=30.6, 95%CI: 3.5-270.6). CONCLUSION: HPV integration might represent a good biomarker of the evolution from HPV infection to cervical cancer. Further prospective studies are required to validate our findings.