Biomedical subjects

Qiulu Pan

Publications and source records attributed to Qiulu Pan.

6 recordsLinked to original sources

PubMed2006

Validation of the 2-DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the Philadelphia (Ph1) chromosome and is present in most of CML patients. The Ph1 chromosome forms a chimeric gene that encodes an abnormal P210 mRNA transcript in most CML patients. Surveillance for minimal residual disease by detection of BCR/ABL transcripts is currently done mostly by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantitation of BCR/ABL transcripts can monitor tumor load and the outcome of therapy. Absolute quantification determines the input copy number of the transcript of interest, usually by plotting the amount of PCR product onto a standard curve based on serial dilutions of the same product cloned in plasmids. Relative quantification describes the change in expression of the target gene in the patient sample relative to that of a control transcript by using the 2-DeltaDeltaCt calculation. The results of real-time RT-PCR for BCR/ABL transcripts are often analyzed by using plasmid DNA standard curves. In the present study, 79 BCR/ABL transcript-positive samples from CML patients who were being monitored for minimal residual disease by real-time quantitative RT-PCR were studied to determine whether the 2-DeltaDeltaCt approach was equivalent to the plasmid standard curve method. BCR/ABL P210 transcripts were quantitated using both the plasmid standard curve method and the 2-DeltaDeltaCt calculation. The comparison of both methods revealed a highly significant and linear correlation between the plasmid standard curve method and the 2-DeltaDeltaCt calculation (R2=0.98, P<0.0001). Furthermore, there was a reduction of preparation time, contamination risk, and reagent usage. The 2-DeltaDeltaCt calculation is a convenient alternative method to derive accurate quantitative information from real time PCR assays.

Data Interpretation, Statistical↗

PubMed2005

KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib.

BACKGROUND: Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in EGFR and KRAS appear to be mutually exclusive. METHODS AND FINDINGS: We sought to determine whether mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug. CONCLUSION: Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both EGFR and KRAS.

Adenocarcinoma↗

PubMed2005

Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas.

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are present in lung adenocarcinomas that respond to the EGFR inhibitors gefitinib and erlotinib. Two types of mutations account for approximately 90% of mutated cases: short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858 (L858R). Screening for these mutations has been based mainly on direct sequencing. We report here the development and validation of polymerase chain reaction-based assays for these two predominant types of EGFR mutations. The assay for exon 19 mutations is based on length analysis of fluorescently labeled polymerase chain reaction products, and the assay for the exon 21 L858R mutation is based on a new Sau96I restriction site created by this mutation. Using serial dilutions of DNAs from lung cancer cell lines harboring either exon 19 or 21 mutations, we detected these mutations in the presence of up to approximately 90% normal DNA. In a test set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases whereas our assays were positive in 29 cases, including 4 cases in which mutations were not apparent by sequencing. These assays offer higher sensitivity and ease of scoring and eliminate the need for sequencing, providing a robust and accessible approach to the rapid identification of most lung cancer patients likely to respond to EGFR inhibitors.

Adenocarcinoma↗

PubMed2005

EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations.

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinase has been extensively studied in breast cancer; however, systematic studies of EGFR gene amplification and protein overexpression in breast carcinoma are lacking. We studied EGFR gene amplification by chromogenic in situ hybridization (CISH) and protein expression by immunohistochemistry in 175 breast carcinomas, using tissue microarrays. Tumors with >5 EGFR gene copies per nucleus were interpreted as positive for gene amplification. Protein overexpression was scored according to standardized criteria originally developed for HER-2. EGFR mRNA levels, as measured by Affymetrix U133 Gene Chip microarray hybridization, were available in 63 of these tumors. HER-2 gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry were also studied. EGFR gene amplification (copy number range: 7-18; median: 12) was detected in 11/175 (6%) tumors, and protein overexpression was found in 13/175 (7%) tumors. Of the 11 tumors, 10 (91%) with gene amplification also showed EGFR protein overexpression (2+ or 3+ by immunohistochemistry). The EGFR mRNA level, based on Affymetrix U133 chip hybridization data, was increased relative to other breast cancer samples in three of the five tumors showing gene amplification. Exons 19 and 21 of EGFR, the sites of hotspot mutations in lung adenocarcinomas, were screened in the 11 EGFR-amplified tumors but no mutations were found. Three of these 11 tumors also showed HER-2 overexpression and gene amplification. Approximately 6% of breast carcinomas show EGFR amplification with EGFR protein overexpression and may be candidates for trials of EGFR-targeted antibodies or small inhibitory molecules.

Adenocarcinoma↗

PubMed2003

Imatinib mesylate in Philadelphia chromosome-positive leukemia of childhood.

BACKGROUND: Initial treatment for adult patients with Philadelphia chromosome-positive (Ph[+]) chronic myelogenous leukemia (CML) now includes imatinib mesylate. However, to our knowledge, there are few data regarding imatinib safety, efficacy, and response monitoring in patients age < 18 years. METHODS: In the current series, the authors report 5 consecutive patients ages 20 months to 12 years with Ph+ leukemia who were treated with imatinib and evaluated for a response using cytogenetics, fluorescent in situ hybridization (FISH), and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on serial bone marrow aspirations. Doses of imatinib were escalated as tolerated from a starting dose of 400 mg/m2 (patients with a body surface area [BSA] < 1 m2) or 400 mg/day (patients with a BSA > 1 m2). RESULTS: After the initiation of imatinib therapy, all 4 patients with CML were found to have no detectable Ph chromosome by cytogenetics (median of 198 days of imatinib therapy; range, 138-346 days), FISH (median of 285 days of imatinib therapy; range, 138-366 days), and real-time RT-PCR (median of 287 days of imatinib therapy; range, 224-366 days). One patient with Ph+ acute mixed lineage leukemia achieved a morphologic disease remission with standard chemotherapy, but within 10 months had increasing Ph positivity in consecutive bone marrow aspirations. Imatinib was added to the intensive leukemia therapy, and within 26 days there were no detectable Ph+ cells in the bone marrow. Mild thrombocytopenia was noted in two patients and transient mild hepatic toxicity was noted in one patient. CONCLUSIONS: Imatinib mesylate was found to be effective in inducing undetectable residual disease in a small cohort of pediatric patients with Ph+ leukemia. Further studies of the use of imatinib in childhood Ph+ malignancies are needed.

Antineoplastic Agents↗

PubMed2003

Relationship between REL amplification, REL function, and clinical and biologic features in diffuse large B-cell lymphomas.

Although it has been suggested that REL is the critical target gene of 2p12-16 amplification in diffuse large B-cell lymphoma (DLBCL), little experimental evidence supports this notion. In the present study, we sought to evaluate the relationship between REL amplification and REL function in a panel of 46 newly diagnosed DLBCLs and to correlate with DLBCL subgroups as identified by gene expression profiles and clinical features. The results indicate that amplification of the REL locus is not associated with accumulation of the active form of REL, as evaluated by immunofluorescence analysis. Upon subgrouping of the DLBCL cases based on gene expression signatures, REL amplification was detected in all subgroups, while high levels of nuclear-located REL were more frequently detected in activated B-cell-like DLBCL. Correlative analyses of REL copy number and REL nuclear accumulation with clinical parameters did not reveal any significant associations. Together these results indicate that 2p12-16 amplification does not lead to abnormal REL activation, suggesting that REL may not be the functional target of the amplification event. Nonetheless, these data indicate that DLBCLs are heterogeneous with respect to REL and thus nuclear factor-kappaB (NF-kappaB) activity.

Adult↗