Performance evaluation comparison of 3 commercially available PCR-based KRAS mutation testing platforms.

The identification of KRAS mutations in patients with certain types of cancer, including colonic adenocarcinoma and non-small cell lung carcinoma, has become increasingly important as these patients are contraindicated from receiving epidermal growth factor receptor-targeted therapies. Several polymerase chain reaction (PCR)-based tests are commercially available for KRAS mutation testing including Applied Biosystems KRAS Mutation Analysis on the ABI3130xl, Qiagen therascreen KRAS RGQ PCR on the Rotor-Gene Q MDx, and Qiagen KRAS Pyro on the PyroMark Q24; however, these tests have not been compared side by side. The purpose of this study was to evaluate the performance characteristics and workflow for 3 PCR-based methods of detecting KRAS mutation status. We evaluated the performance characteristics and workflow for 3 commercially available KRAS mutation detection platforms. All of the 188 samples run were successful, with 29% being positive for the KRAS mutation. Of the positive tests, Applied Biosystems detected 84% of the positive cases, whereas Qiagen therascreen RGQ and Qiagen KRAS Pyro detected 100% of the positive cases. In cases of discrepancy between Applied Biosystems and therascreen RGQ, Pyro agreed with therascreen RGQ 95% of the time. Qiagen therascreen RGQ and Pyro were comparable in terms of sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, with all values being 100%. All 3 techniques accurately identified the appropriate mutation in the known control specimens. In summary, all 3 tests are relatively comparable for detecting the KRAS mutation, with Applied Biosystems having a slightly lower sensitivity, negative predictive value, and accuracy than therascreen RGQ and Pyro.

Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung.

Cell-transfer technique has been proven useful for performing immunocytochemistry on fine-needle aspiration smears. However, its utility for EGFR and KRAS molecular testing has not been validated. Molecular testing was performed using the cell-transfer technique on both Papanicolaou-stained ethanol-fixed and Hema 3-stained air-dried smears from 32 fine-needle aspiration samples that had diagnoses of adenocarcinoma of the lung, and then was compared to the results of the corresponding formalin-fixed paraffin-embedded tissues. The molecular testing was successfully performed on 32 of 32 ethanol-fixed and 31 of 32 air-dried samples. The molecular results on ethanol-fixed and air-dried smears showed 100% agreement. There is 100% (32/32) agreement for the EGFR and 97% (31/32) agreement for the KRAS between the cell-transfer technique and formalin-fixed paraffin-embedded tissues. One discrepant case was due to low percentage of tumor cells on the smears. Cell-transfer technique is a reliable alternative method for EGFR and KRAS testing if the cell blocks lack adequate cellularity.

KRAS mutation is present in a small subset of primary urinary bladder adenocarcinomas.

AIMS: To determine whether KRAS mutations occur in primary bladder adenocarcinoma. METHODS AND RESULTS: Twenty-six cases of primary urinary bladder adenocarcinoma were analysed. DNA was extracted from formalin-fixed, paraffin-embedded tissue and amplified with shifted termination assay technology, which recognizes wild-type or mutant target sequences and selectively extends detection primers with labelled nucleotides. A mutation in KRAS was found in three (11.5%) of 26 primary bladder adenocarcinomas. Two of these three cases exhibited a G13D mutation, whereas the remaining case contained a mutation in G12V. None of the ten cases of urothelial carcinoma with glandular differentiation displayed KRAS mutation. Colonic adenocarcinoma contained a KRAS mutation in 18 (33%) of 55 cases. There was no distinct difference with regard to grade, stage or outcome according to the limited clinicopathological data available. However, the two youngest patients, aged 32 and 39 years, in our study group, with a mean population age of 61 years, were found to have mutations in KRAS. CONCLUSIONS: KRAS mutations are present in a small subset of primary urinary bladder adenocarcinomas. Future clinical trials for treatment of bladder adenocarcinoma, employing targeted therapies similar to those used for treatment of colon cancer, may also benefit from the predictive implications of KRAS mutational testing.

Identification of a domain in the V0 subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase.

Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.

Mutational analysis of the stator subunit E of the yeast V-ATPase.

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.