Qiulu Pan

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.

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.

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.

Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia

Minimal residual disease (MRD) is an independent prognostic factor in childhood acute lymphoblastic leukemia (ALL). The most widely applied MRD assays in ALL are flow cytometric identification of leukemia immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. We measured MRD by both assays in 227 patients with childhood B-lineage ALL. Of 1375 samples (736 bone marrow and 639 peripheral blood) examined, MRD was <0.01% in 1200, and ⩾0.01% in 129 by both assays; MRD levels measured by the two methods correlated well. Of the remaining 46 samples, 28 had MRD ⩾0.01% by flow cytometry but <0.01% by PCR. However, PCR (which had a consistent sensitivity of 0.001%) detected leukemic gene rearrangements in 26 of these 28 samples. Conversely, in 18 samples, MRD was ⩾0.01% by PCR but <0.01% by flow cytometry. In nine of these samples, flow cytometry had a sensitivity of 0.001%, and detected aberrant immunophenotypes in eight samples. Therefore, the two most widely used methods for MRD detection in ALL yield concordant results in the vast majority of cases, although the estimated levels of MRD may vary in some. The use of the two methods in tandem ensures MRD monitoring in all patients.

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia

Children with acute lymphoblastic leukemia (ALL) with 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 104 or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels ≥ 1 in 104. The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels <1 in 104. In only two samples results were discordant: 2 in 104 and 5 in 104 leukemic cells by PCR but <1 in 104 by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.

Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia

Minimal residual disease (MRD) at the end of remission-induction therapy predicts relapse in acute lymphoblastic leukemia (ALL). We examined the clinical significance of levels below the usual threshold value for MRD positivity (0.01%) in 455 children with B-lineage ALL, using polymerase chain reaction amplification of antigen-receptor genes capable of detecting at least 1 leukemic cell per 100 000 normal mononucleated cells (0.001%). Of the 455 clinical samples studied on day 46 of therapy, 139 (30.5%) had MRD 0.001% or more with 63 of these (45.3%) showing levels of 0.001% to less than 0.01%, whereas 316 (69.5%) had levels that were either less than 0.001% or undetectable. MRD measurements of 0.001% to less than 0.01% were not significantly related to presenting characteristics but were associated with a poorer leukemia cell clearance on day 19 of remission induction therapy. Patients with this low level of MRD had a 12.7% (+/- 5.1%; SE) cumulative risk of relapse at 5 years, compared with 5.0% (+/- 1.5%) for those with lower or undetectable MRD (P < .047). Thus, low levels of MRD (0.001%-< 0.01%) at the end of remission induction therapy have prognostic significance in childhood ALL, suggesting that patients with this finding should be monitored closely for adverse events.

Both Gene Amplification and Allelic Loss Occur at 14q13.3 in Lung Cancer

Purpose: Because loss of Nkx2-8 increases lung cancer in the mouse, we studied suppressive mechanisms in human lung cancer. Experimental Design:NKX2-8 is located within 14q13.3, adjacent to its close relative TTF1/NKX2-1. We first analyzed LOH of 14q13.3 in forty-five matched human lung cancer and control specimens. DNA from tumors with LOH was then analyzed with high-density single-nucleotide polymorphism (SNP) arrays. For correlation with this genetic analysis, we quantified expression of Nkx2-8 and TTF1 mRNA in tumors. Finally, suppressive function of Nkx2-8 was assessed via colony formation assays in five lung cancer cell lines. Results: Thirteen of forty-five (29%) tumors had LOH. In six tumors, most adenocarcinomas, LOH was caused by gene amplification. The 0.8-Mb common region of amplification included MBIP, SFTA, TTF1, NKX2-8, and PAX9. In 4 squamous or adenosquamous cancers, LOH was caused by deletion. In three other tumors, LOH resulted from whole chromosome mechanisms (14−, 14+, or aneuploidy). The 1.2-Mb common region of deletion included MBIP, SFTA, TTF1, NKX2-8, PAX9, SLC25A21, and MIPOL1. Most tumors had low expression of Nkx2-8. Nevertheless, sequencing did not show NKX2-8 mutations that could explain the low expression. TTF1 overexpression, in contrast, was common and usually independent of Nkx2-8 expression. Finally, stable transfection of Nkx2-8 selectively inhibited growth of H522 lung cancer cells. Conclusions: 14q13.3, which contains NKX2-8, is subject to both amplification and deletion in lung cancer. Most tumors have low expression of Nkx2-8, and its expression can inhibit growth of some lung cancer cells. Clin Cancer Res; 17(4); 690–9. ©2010 AACR.

Quantification of minimal residual disease in T-lineage acute lymphoblastic leukemia with the TAL-1 deletion using a standardized real-time PCR assay

Hematologic relapse remains the greatest obstacle to the cure of children with acute lymphoblastic leukemia (ALL). Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current PCR methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, we developed a real-time quantitative PCR (RQ-PCR) assay for detection of leukemic cells that harbor the TAL-1 deletion. We studied serial dilutions of leukemic DNA and found the assay had a sensitivity of detection of one leukemic cell among 100 000 normal cells. We then investigated 23 samples from eight children with ALL in clinical remission. We quantified residual leukemic cells by using the TAL-1 RQ-PCR assay and by using limiting dilution analysis. In 17 samples, both methods detected MRD levels ⩾0.001%. The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.926). In the remaining six samples, both methods detected fewer than 0.001% leukemic cells. We conclude the TAL-1 RQ-PCR assay can be used for rapid, sensitive and accurate assessment of MRD in T-lineage ALL with the TAL-1 deletion.

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

Detecting clonal T-cell receptor (TCR)-gamma gene rearrangements (GRs) is an important adjunct test for diagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol), which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V), with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-gamma GR. The combination of TCR-beta, mono-V TCR-gamma and multi-V TCR-gamma detected more clonal cases (68/144, 47%) than any individual PCR assay. We detected clonal TCR-beta GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-gamma primers, the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-gamma primers improved the sensitivity for detecting clonality, 60/68 (88%). Combining either mono-V or multi-V TCR-gamma primers with TCR-beta primers also improved the sensitivity, 64/68 (94%). Significantly, TCR-gamma V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.

Comparative evaluation of three JAK2V617F mutation detection methods

The correlation of JAK2V617F with a proportion of chronic myeloproliferative disorders has generated numerous studies focused on the development of molecular-based assays for JAK2V617F detection. The current parallel study comparatively evaluated 3 JAK2V617F molecular detection methods. Genomic DNA from blood or bone marrow was assayed by 3 laboratories using allele-specific polymerase chain reaction (AS-PCR) or kit-based restriction fragment length polymorphism methods, which used polyacrylamide gel or capillary electrophoresis analysis. In addition, samples were sequenced in 2 of the laboratories. Results found 100% concordance among the 3 methods, with analytic sensitivities of 5% for both kit methods and 0.01% for AS-PCR. The kitbased assays detect JAK2V617F with equal sensitivity regardless of analysis method, and, despite greater sensitivity of AS-PCR, all 3 methods yielded 100% concordant results for this 36-sample set. Consistent with other reports, direct sequencing was insufficiently sensitive to serve as an initial diagnostic tool for JAK2V617F detection.