The application and diagnostic utility of immunocytochemistry on direct smears in the diagnosis of pulmonary adenocarcinoma and squamous cell carcinoma.

The importance of subclassifying pulmonary nonsmall cell carcinoma (NSCLC) in cytologic material is becoming increasingly paramount. Occasionally, cell blocks traditionally used for ancillary studies are sparsely cellular or acellular. Hence, we investigated the diagnostic utility of immunocytochemistry for Napsin-A, TTF-1, and p63 on direct smears of NSCLC. Immunohistochemistry for Napsin-A was initially tested on a tissue microarray (TMA) composed of pulmonary adenocarcinoma. Subsequently, in 25 cases, immunocytochemistry for Napsin-A, TTF-1, and p63 was performed on cytologic direct smears. Smears were prepared from tumor cells scraped from lung resection specimens (n = 10), endobronchial ultrasound-guided transbronchial fine-needle aspirates (n = 13), and pelleted cell material from pleural effusions (n = 2). Immunohistochemistry utilizing the TMA revealed Napsin-A positivity in 73% of pulmonary ADCs. Next, immunocytochemistry on direct cytologic smears demonstrated a Napsin-A(+)/TTF-1(+) immunophenotype in 15 of 18 adenocarcinomas; p63 was completely negative (n = 12) or only focally positive (n = 3) in these 15 adenocarcinomas. The remaining three adenocarcinomas were negative for all three markers. All six squamous cell carcinomas were Napsin-A(-)/TTF-1(-) and diffusely p63(+). In conclusion, direct smears represent a feasible and robust source of cellular material for immunocytochemical studies to diagnose pulmonary ADC and SQC. Our method allows the cytologist to confirm on site that material for diagnostic immunocytochemistry is present thereby serving as a safeguard in instances where the cell block is of insufficient cellularity.

Severe gammaherpesvirus-induced pneumonitis and fibrosis in syngeneic bone marrow transplant mice is related to effects of transforming growth factor-ß.

Pulmonary infections and pneumonitis occur frequently after hematopoietic stem cell transplantation. Using a syngeneic mouse model of bone marrow transplantation (BMT), we have previously demonstrated that BMT mice are more susceptible to acute gammaherpesvirus 68 (MHV-68) replication at day 7 after infection. By day 21, the virus is latent in lungs of BMT and control mice, and there is no difference in viral load. Despite similar latent viral load, BMT mice develop severe pneumonitis associated with reduced oxygen saturation, fibrosis, peripheral inflammation, hyaline membranes, and foamy alveolar macrophages, a phenotype that persists for 7 weeks after infection. BMT mice demonstrate increased bronchoalveolar lavage (BAL) cells, and this population is enriched in neutrophils and T cells. Alternatively, activated macrophages appear earlier than do classically activated macrophages. BAL fluid from BMT mice at day 21 after infection contains increased levels of hydrogen peroxide, nitrite, and transforming growth factor-ß (TGF-ß). Mice expressing the dominant-negative transgene dn-TGFßRII in multiple cell types were used as BMT donors. BMT mice with T-cell dnTGFßRII are largely protected from the pneumonitis phenotype, whereas mice with CD11c-dnTGFßRII BMT mice are only modestly protected from pneumonitis. Protection in BMT mice with T-cell dnTGFßRII is associated with decreased TGF-ß derived from parenchymal cells in the BAL fluid, lower nitrite levels, and reduced apoptosis, whereas alternatively activated macrophage markers are unchanged.

The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to stained cytologic smears of lung carcinoma.

EGFR and KRAS mutation analyses are of increasing importance for guiding the treatment of non-small cell lung carcinomas. Insufficient cellularity of cell blocks can represent an impediment to the performance of these tests. We investigated the usefulness of cytologic direct smears as an alternative specimen source for mutation testing. Tumor cell-enriched areas from freshly prepared and archived rapid Romanowsky-stained direct smears in 33 cases of lung carcinoma were microdissected for DNA isolation and evaluated for EGFR and KRAS mutations. EGFR mutations were detected in 3 adenocarcinomas; 2 tumors had the L858R substitution and 1 an exon 19 deletion. KRAS mutations affecting codon 12, 13, or 61 were detected in 11 cases (8 adenocarcinomas and 3 non-small cell carcinomas). EGFR and KRAS mutations were mutually exclusive. Hence, archived and freshly prepared direct smears represent a robust and valuable specimen source for molecular studies, especially when cell blocks exhibit insufficient cellularity.

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.