Mariona Llatjós

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.

A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells

Background: Detection of epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients has relied on DNA purification from biopsies, amplification, and sequencing. However, the number of tumor cells in a sample is often insufficient for EGFR assessment. Methods: We prospectively screened 1380 NSCLC patients for EGFR mutations but found that 268 were not evaluable because of insufficient tumor tissue. We therefore developed and validated a method of detecting EGFR mutations in these samples. Tumor cells were microdissected into polymerase chain reaction buffer and amplified. EGFR mutations were detected by length analysis of fluorescently labeled polymerase chain reaction products and TaqMan assay. Results: We determined EGFR status in 217 (81%) of the 268 primary NSCLC samples not evaluable in our original study—fresh and paraffin-embedded with less than 150 cells. Exon 19 deletions were detected in 11.5% of patients and exon 21 L858R mutations in 5.5%. In addition, the exon 20 T790M mutation was detected in 6 of 15 (40%) patients at the time of progression to erlotinib. The primary, sensitive mutation was present in all tumor cells, whereas the T790M mutation was absent in some groups. Conclusions: The method presented here eliminates the need for DNA purification and allows for detection of EGFR mutations in samples containing as few as eight cancer cells.

Intraoperative assessment of sentinel lymph nodes in patients with breast carcinoma: Accuracy of rapid imprint cytology compared with definitive histologic workup

As sentinel lymph node biopsy (SNB) becomes a new surgical standard in the treatment of patients with breast carcinoma, there is an emergent need for a fast and accurate method with which to assess the SN intraoperatively, so a decision can be made regarding whether to perform axillary lymph node dissection during primary surgery. In the current study, the authors performed a prospective investigation of the relative merits of imprint cytology for that purpose.

High prevalence of human papillomavirus infection in the anus, penis and mouth in HIV-positive men.

Human papillomavirus (HPV) types are associated with squamous cell cancers. HIV infection is linked with a higher prevalence of anal HPV infection. It is important to assess whether HPV is present in other body parts involved in sexual practices to establish a cancer prevention program. A high prevalence of high-risk HPV types was present in the anus, penis and mouth (78, 36 and 30%, respectively) in a cohort of HIV-infected males (men who have sex with men and heterosexual), without evidence of pathology in these areas.

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

Information about a tissues 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.

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

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.

Epidemiological data of different human papillomavirus genotypes in cervical specimens of HIV-1-infected women without history of cervical pathology.

Aim:To study the epidemiology of different human papillomavirus (HPV) genotypes in cervical samples of HIV-1-infected women with normal Papanicolau smears. Design:Retrospective analysis of a prospective cohort. Patients and Methods:We selected HIV-1-infected women with 2 consecutive normal Papanicolau smears at baseline and at least 1 baseline and 1 follow-up cervical sample. HPV infection was assessed by second-generation hybrid capture (HC-2) and multiplex polymerase chain reaction (mPCR). HPV genotypes were determined by mPCR. Results:From a cohort of 139 women followed up to 4 years, 93 women meeting the inclusion criteria were analyzed. The mean period between samples was 20 months (range, 6-44 months). HPV baseline prevalence was 63% [59/93; 95% confidence interval (CI), 53% to 73%] using polymerase chain reaction and 41% (38/93; 95% CI, 31% to 51%) using HC-2, P = 0.007 (kappa, 0.45; P = 0.001). The most prevalent high oncogenic risk genotypes (HR-HPV) were HPV-16 (28%), HPV-33 (18%), HPV-52 (12%), HPV-58 (11%), and HPV-39 (11%). Infection with multiple HPV genotypes was detected in >40% of women. HPV infection persisted at follow-up in 86% (51/59; 95% CI, 77% to 95%) by polymerase chain reaction and 76% (29/38; 95% CI, 62% to 90%) by HC-2. HPV infection persisted in 55% of women with samples available beyond 3 years. The actuarial probabilities of clearance and incidence of HPV infection at 36 months were 16% and 45%, respectively. Conclusions:HPV infection is highly prevalent and persistent among HIV-1-infected women with normal Papanicolau smears. HR-HPV genotypes other than HPV-16 (HPV-33, HPV-52) are frequently detected in HIV-infected women. mPCR provides better surveillance of HPV infection than HC-2 methods.

Cytology of bronchial biopsy rinse fluid to improve the diagnostic yield for lung cancer

Sampling techniques are combined during bronchoscopy to increase the diagnostic yield for endobronchial malignant tumours. Bronchial biopsy provides the definitive histological diagnosis in most cases, but accompanying cytological procedures such as washing, brushing, needle aspiration or imprint cytology can increase diagnostic yield. In this prospective study, a different cytological technique, that could enhance the diagnostic yield of bronchoscopy without increasing its time or cost, was tested. Flexible bronchoscopy was performed in 93 patients suspected of having pulmonary neoplasms. Bronchial biopsies were initially placed in a balanced salt solution. When bronchoscopy was finished, all visible tissue fragments were removed and placed in formalin to undergo histopathological examination and the rinse fluid was sent for cytological examination. Washing was performed routinely but no cytological brushing was employed. Eighty-two patients had final diagnoses of malignant neoplasm. In four (4.8%) of these patients, the only positive result came from the cytological examination of the bronchial biopsy rinse fluid. No false-positive results were found. The agreement with the histological results was 81.8%. The addition of bronchial biopsy rinse-fluid examination increased the sensitivity of bronchoscopy from 65.8% to 70.7% (McNemars p=0.009). The cytological study of bronchial biopsy rinse fluid offers reliable positive results in an additional 4.8% of cases, thus enhancing bronchoscopic diagnostic yield for malignant endobronchial tumours while neither prolonging the procedure nor increasing costs.

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.

Frecuencia y riesgo de neoplasia broncopulmonar relacionada con asbesto

Objetivo Determinar la prevalencia de neoplasia broncopulmonar relacionada con el asbesto y la importancia de la exposicion laboral a esta fibra inorganica como factor de riesgo. Pacientes y metodo Se realizo un estudio transversal de 82 pacientes con neoplasia broncopulmonar (edad media [DE] 62 [9] anos) y 53 pacientes sin enfermedad pleuropulmonar (edadmedia [DE] 63 [13] anos), con identificacion de la exposicion laboral a asbesto por medio de cuestionario. Se cuantifico la concentracion de cuerpos de asbesto (CA) en lavado broncoalveolar (BAL) (93 pacientes) o tejido pulmonar (42 pacientes) despues del procesado de las muestras por digestion quimica, expresando los resultados como CA/ml BAL o CA/g tejido pulmonar seco. Una concentracion de CA superior a 1 CA/ml o 1.000 CA/g fue considerada como marcador de una concentracion elevada de asbesto en tejido pulmonar, potencialmente causante de enfermedad pleuropulmonar. La importancia de la exposicion laboral a asbesto como factor de riesgo de neoplasia broncopulmonar se determino con modelos de regresion logistica. Resultados En el grupo con neoplasia broncopulmonar, 25 pacientes reconocian haber tenido contacto laboral con asbesto (30%) y en 13 se detectaron CA en BAL o en tejido pulmonar (24%), con una concentracion elevada en 3 casos (4%). En el grupo sin enfermedad pleuropulmonar, 6 pacientes reconocian haber tenido contacto con asbesto (11%) y en 13 se detecto la presencia de CA en alguna de las muestras estudiadas (24%), en ningun caso se hallaron concentraciones elevadas. En los analisis univariantes por regresion logistica el diagnostico de neoplasia broncopulmonar se asociaba a tabaquismo (odds ratio 10,10; intervalo de confianza [IC] del 95%: 3,50-29,13) y a contacto laboral con asbesto (odds ratio 3,69; IC del 95%: 1,39-9,77). Esta ultima relacion persistia como estadisticamente significativa al ajustar el modelo logistico para tabaquismo (odds ratio 2,80; IC del 95%: 1,00-7,84). Conclusiones En Espana un 4% de las neoplasias broncopulmonares estan relacionadas con el asbesto actuando en sinergia con el tabaquismo. La exposicion laboral a esta fibra inorganica duplica el riesgo de padecer este tipo de neoplasia.