Dear Sir,
Lung cancer causes the most cancer-related deaths in western countries and its treatment remains a challenge. Encouraging results have been obtained recently with new chemotherapeutic agents such as paclitaxel in non-small-cell lung cancer. A striking association, however, has been reported between chemoresistance of advanced-stage cancer to taxanes, poor prognosis and the presence of somatic mutations in the -tubulin class I (TUBB) gene in non-small-cell lung cancer. 1 We attempt to confirm this finding in an independent series of patients with earlier stage disease. This would be important for selection of potential adjuvant chemotherapy.
Paclitaxel treatment blocks the cell cycle by disrupting the function of microtubules, 2 which are an integral part of a variety of cell structures, such as the mitotic apparatus, cytoskeleton and cilia. Microtubules are heterodimers composed of ␣and -tubulin. The -tubulins are a multigene family of 15-20 members with at least 4 main functional isotypes, Class I, II, III, IVa and IVb and several pseudogenes. 3,4 Class I, II, III and IVb isotypes are expressed in the lung, although at varying levels. 5 We studied the genomic sequence of Class I -tubulin from a series of 29 patients with resected lung tumors (15 male, 14 female, median age 67 years). Histological type was adenocarcinoma (8), squamous cell (9 cases), large cell (6), bronchioalveolar (1) carcinoid/neuroendocrine (4) and mucoepidermoid carcinoma (1). Stage was IA in 10, IB in 10 and IIIA in 3 cases. No patient had received chemotherapy. Archival pathology specimens were re-evaluated for pathology and tumor and normal tissue was manually microdissected from H&E-stained serial sections. DNA was purified using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN).
The section of exon 4 (codons 93-289) analyzed previously by Monzo et al. 1 was PCR amplified in 3 overlapping fragments. Fragment A was amplified using primers designed in this laboratory, from GenBank sequence J00314; 5Ј-TTC TCC TGA CTG GCA TTC CAG GT-3Ј (forward strand) and 5Ј-CTC GTT GTC AAT GCA ATA GGT-3Ј (reverse strand). The forward primer contained mostly intronic sequence. Fragment B and C were amplified using primer sequences 1 published previously, but the orientation of the Fragment C reverse primer was corrected. PCR products were amplified using AmpliTaq Gold (Applied Biosystems, Foster City, CA) in the following conditions: Fragment A, 94°C 30 sec, 60°C 30 sec,