Mutations In Tumor Tissue Research Articles

HLA typing by next‐generation sequencing – getting closer to reality

Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer

KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer

Abstract S4-05: Exome sequencing identifies shift in TP53 and PIK3CA mutation status after paclitaxel-based neoadjuvant chemotherapy in breast cancer

Abstract Purpose: The changed gene-expression profiles of breast cancer under neoadjuvant chemotherapy (NCT) have been described. However, it remains unclear whether chemotherapy affects somatic mutation status in breast cancer. We investigated the influence of paclitaxel-based chemotherapy on somatic mutations in tumor tissues from patients with breast cancer. Patients and Methods: Samples were derived from two cohorts: first, 296 patients who underwent paclitaxel-based NCT with matched pre- and post-chemotherapy tumor tissues; second, 81 patients who underwent paclitaxel-based chemotherapy with pre-chemotherapy tumor tissues. First, we performed whole exome sequencing (WES) to examine the exomes (100-fold coverage) of two paired basal-like breast cancer samples (pre-treatment tumor biopsies and post-treatment tumors). Shift in somatic mutation status was validated by Sanger sequencing and analyzed in an extension cohort of 296 patients. Furthermore, we performed microdissection and genetic heterogeneity analysis for samples from 81 patients who underwent paclitaxel-based chemotherapy. Somatic mutation status was determined to reveal potential impact of paclitaxel-based chemotherapy on mutation status. Results: Compared with post-treatment biopsies, the analysis of WES data identified and validated TP53 and PIK3CA — as significantly mutated genes in pre-treatment tumors with a mutation frequency of approximately 10% in an independent extension cohort of 296 paired samples of different molecular subtypes. Somatic mutations of TP53 or PIK3CA were detected in 24.3% of the pre-chemotherapy tumor samples (35 of 296 for TP53, 32 of 296 for PIK3CA, 5 of 296 for both genes, respectively) but in only 12.2% of the post-chemotherapy tumor samples (18 of 296 for TP53, 16 of 296 for PIK3CA, 2 of 296 for both genes, respectively). The decrease in mutation rate was statistically significant (P < 0.001). Patients with TP53/PIK3CA mutations switched from positive to negative after chemotherapy had better partial response than patients with no change or with a reverse change (P = .008). Furthermore, patients with TP53/PIK3CA mutations switched from positive to negative had improved disease-free survival (P = 0.018) and improved overall survival (P = 0.032) relative to patients with no change or with a reverse change. In the second cohort, 18.5% of the tumors (15 of 81) showed intratumoral heterogeneity of TP53 or PIK3CA somatic mutations, whereas 81.5% (66 of 81) were homogeneous, either with mutations of certain genes or not. Conclusion: Our data reveal the novel concept that significant evolution might occur during chemotherapy. Chemotherapy may reduce somatic mutation frequency in patients with breast cancer, likely the result of a preferential response of highly fit sub-clones without mutations of certain genes. In addition, the identification of somatic mutations of TP53 and PIK3CA as biomarkers for prediction of treatment response and prognosis might help us optimize choice for sequential therapy and improve patients’ survival. KEY WORDS: Exome sequencing; TP53; PIK3CA; neoadjuvant chemotherapy; breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S4-05.

Abstract C100: KRAS mutations in circulating cell-free DNA of colorectal cancer patients in progression after MoAb anti-EGFR therapy.

Abstract Background: Anti-EGFR therapy is a standard treatment strategy in patients with metastatic colorectal cancer (mCRC) with a wild type KRAS status. Inevitably, resistance mechanisms arise in the majority of patients during treatment, and re-biopsy of the tumor is the only way to characterize the molecular alterations responsible for the resistance. Circulating cell-free tumor DNA (cftDNA) is released into the bloodstream by tumor cells, and its recovery could represent a valid non-invasive alternative to biopsy for the molecular characterization of the tumor. The aim of this study is to search for KRAS mutations in the cftDNA of mCRC patients in progression after treatment with an anti-EGFR MoAb. Methods: Fourteen mCRC patients with KRAS wild type status identified previously in tumor tissue by pyrosequencing were treated with an anti-EGFR MoAb until progression. cftDNA was extracted from 1-2 ml of plasma collected at baseline and after progression. Digital Real Time PCR was performed for KRAS G12D and G12V mutations using a QX100™ ddPCR™ System (Bio-Rad). Samples were then loaded into a droplet reader that discriminates between the different fluorescence amplitudes on the basis of target gene amplification. Results: KRAS mutation analysis performed by Digital PCR revealed that mutations (2 G12V and 1 G12D) were present in cftDNA extracted after progression in 3 (21%) of the 14 patients. KRAS analysis was then also performed in DNA extracted from primary tumor tissue and from basal cftDNA. Interestingly, the same mutations were found in the corresponding tumor tissue, confirming the higher sensitivity of Digital PCR with respect to pyrosequencing analysis. Moreover, in 2 of the 3 patients, the KRAS mutation was also present in basal cftDNA. Discussion: Our preliminary results reveal that cftDNA could be a suitable non-invasive approach for the molecular characterization of tumors and could be especially useful to monitor the development of resistance mechanisms, thus avoiding the need for re-biopsy. We also showed that KRAS mutations would not seem to represent a mechanism of acquired resistance to anti-EGFR MoAbs in mCRC patients and are probably present in minor clones at baseline but not detected by standard methods. The use of highly sensitive methods, such as Digital PCR, could enable us to detect these minor mutations in tumor tissue and to monitor response to anti-EGFR MoAbs using cftDNA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C100. Citation Format: Paola Ulivi, Marzia Del Re, Alessandro Passardi, Giorgia Marisi, Dino Amadori, Fabio Casacci, Martina Valgiusti, Wainer Zoli, Romano Danesi. KRAS mutations in circulating cell-free DNA of colorectal cancer patients in progression after MoAb anti-EGFR therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C100.

Comparison of different methods for detecting epidermal growth factor receptor mutations in peripheral blood and tumor tissue of non-small cell lung cancer as a predictor of response to gefitinib

BackgroundPrevious studies have reported that epidermal growth factor receptor (EGFR) mutation in tumor tissue and peripheral blood can predict the response to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). However, the heterogeneity of the sample sources makes it difficult to evaluate the detecting methodologies. The goal of this study is to compare different methods for analyzing EGFR mutation in blood and tumor tissue.Materials and methodsFifty-one advanced NSCLC patients treated with gefitinib were included in the study. The EGFR mutation status of each patients’ blood was analyzed by denaturing high-performance liquid chromatography (DHPLC), mutant-enriched liquidchip (ME-Liquidchip), and Scorpion Amplification Refractory Mutation System (Scorpion-ARMS) kits. EGFR mutation information in paired tumor samples detected by Scorpion-ARMS served as a reference. Comparative analyses were performed on mutation status results obtained from different methods and on the association between the clinical outcome of TKI treatment and EGFR mutation status.ResultsThe response rate (RR) in the whole group was 33.3%. EGFR mutation rates were identified as 15.7%, 27.5%, and 29.4% by DHPLC, ME-Liquidchip, and Scorpion-ARMS in blood, respectively. In 34 cases that had paired tumor samples, the mutation rate in tissue was 41.2%. The RRs of patients with mutation detected by different methods were 71.4% (tumor), 62.5% (blood, DHPLC), 50.0% (blood, ME-Liquidchip), and 66.7% (blood, Scorpion-ARMS). EGFR mutation detected by Scorpion-ARMS in blood and tumor tissues had better prediction of RR to EGFR-TKI (P = 0.002 and P = 0.001) than mutation detected with DHPLC and ME-Liquidchip.ConclusionTumor tissue sample is the best source for EGFR mutation analysis in NSCLC patients. Peripheral blood samples may be used as an alternative source only in special conditions. Scorpion-ARMS, DHPLC, or ME-Liquidchip methods are all optional for detecting tumor EGFR mutation from blood.

Survey of KRAS, BRAF and PIK3CA mutational status in 209 consecutive Italian colorectal cancer patients

Molecular testing for KRAS and BRAF mutations in tumor tissue is a fundamental tool to identify patients with metastatic colorectal cancer (CRC) who are eligible for anti-EGFR monoclonal antibody therapy. We here report a molecular analysis by high-resolution melting analysis and direct sequencing of KRAS, BRAF and PIK3CA hot spot mutations in 209 Italian CRC patients. One hundred and ten patients (51%) were identified who were potentially nonresponders to anti-EGFR therapy: 90/209 patients (43%) harboring KRAS mutations, 13/117 (11.1%) with the V600E BRAF mutation, and 7/209 (3.3%) with mutations in PIK3CA exon 20. The prevalence of BRAF and PIK3CA mutations was significantly higher in patients older than 65 years (p=0.014 and p=0.018), while patients with triple-negative tumors were significantly younger than mutation carriers (p=0.000011). Patients with gene mutations also showed a trend towards preferential tumor location in the colon (p=0.026). Moreover, although involving a relatively small number of samples, we report the presence of a discordant mutational profile between primary tumors and secondary lesions (3/9 patients), suggesting that it is worthwhile to test other available tissues in order to better define the efficacy of targeted therapy. Further correlations of specific clinical features with tumor mutational profile could be helpful to predict the response of CRC patients to monoclonal antibody therapy.

Detection of EGFR and KRAS somatic mutations in tumor tissue and peripheral blood by a liquidchip technology for patients with advanced non-small cell lung cancer.

e18142 Background: Responsiveness of non-small cell lung cancer (NSCLC) to EGFR tyrosine kinase inhibitors has been confirmed to relate to mutations in the epidermal growth factor receptor (EGFR) signaling pathway genes. This study’s aim is to detect mutations in two important components of the EGFR signaling pathway, EGFR and KRAS, using a novel liquidchip technology, and to evaluate the correspondence between tumor and plasma DNA in the same patient. Methods: 58 advanced primary NSCLC ( IIIB or ‡W) tumor tissues and matched peripheral blood samples (MPBS) were obtained. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were performed after DNA extraction. A novel liquidchip platform for simultaneous detection multiple alleles of DNA somatic mutations was applied to detect the EGFR and KRAS status in matched samples. Results: EGFR exon 19 and exon 21 mutation rate was detected to be 29.3% (17/58) and 17.2% (10/58) respectively in tumor tissues, while 20.7% (12/58) and 12.1% (7/58) in MPBS. Among those 27 patients who harbor either EGFR exon 19 or exon 21 mutations in tissues, 19 (70.4%) of them were detected to have the same mutations in their peripheral blood samples. EGFR exon 20 T790M mutations were found in one of the paired samples. All the patients who were detected to have wild-type EGFR in tissues were detected to have wild-type in MPBS as well. In 50 of 58 (86.2%) of the paired samples, the EGFR mutation status in the tumors was consistent with those in the MPBS. The KRAS exon 2 mutation was also found in both blood and tumor samples in one patient; however only in the blood of another patient, not in his tumor. Conclusions: The liquidchip technology may serve as an effective method for detecting multiple gene mutations. EGFR and KRAS gene mutations in blood DNA were highly consistent with the corresponding tissue samples, and therefore, peripheral blood could be used as an alternative sample to predict patients’ response to the anti-EGFR targeted therapy. Further studies to evaluate the clinical application are suggested.

Abstract 962: Multiplexed mutational profiling of breast tumor as an efficient clinical tool for personalized breast cancer treatment

Abstract Introduction: Breast cancer is increasingly being recognized as a heterogeneous disease and previous studies have reported various oncogenic mutations in breast cancer specimens. However, a validated methodology for clinical assessment of mutation profiles is lacking. Applying such technologies will be increasingly important to accelerate development of targeted therapies toward tumor-specific oncogene pathways. Utilizing a high-throughput genotyping platform we evaluated the commonly described somatic mutations in tumor tissue among women diagnosed with metastatic breast cancer. Methods: We developed a CLIA (Clinical Laboratory Improvement Amendments) validated, highly sensitive multiplexed PCR-based assay (SNaPshot) to simultaneously identify 130 previously described mutations in 13 cancer genes using DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue. Data regarding demographics, tumor phenotype based on estrogen and progesterone receptor (ER, PR) status, HER-2 (Human Epidermal growth factor Receptor-2) status, BRCA carrier status, and clinical outcomes, was abstracted from clinical charts. Results: A total of 138 tumors from women with metastatic breast cancer, median age 69 (range 29-82), were analyzed. In this study, 34.7% of tumors had a detectable mutation, with PIK3CA mutation (70.8%) being the most common mutation detected followed by TP53 (27.1%), and Akt (4%). Unanticipated mutations in KRAS and BRAF were observed at lower frequencies. Detectable mutations were seen in all tumor sub-types although the frequency varied by tumor phenotype: ER+/PR+ (34.5%), ER+/PR- (52.6%), HER-2+ (44.4%), and triple negative (23.2%) tumors. Similarly, while the type of mutation varied somewhat by tumor phenotype, the common driver mutations were seen in all tumor subtypes. For PIK3CA mutations: ER+/PR+ (27.7%), ER+/PR- (42.8%), HER-2+ (29.6%), triple negative (6.7%) tumors, and for TP53 mutations: ER+/PR+ (3.6%), ER+/PR- (14.2%), HER-2+ (18.5%) and triple negative (10%) tumors. Correlation with clinical outcomes is ongoing. BRCA1/2 carriers were less likely to have a detectable somatic mutation in their tumor (15.3%) than patients with negative BRCA1/2 status (25.8%). Only 4 patients (2.8%) had more than one detectable somatic mutation. Conclusion: Oncogenic driver mutations can be detected by SNaPshot from routinely-collected FFPE tissue specimens obtained from women seen in the oncology clinic for metastatic breast cancer. While the type and frequency of mutation varies somewhat by tumor phenotype, the common oncogenic mutations are seen in all breast cancer subtypes and are often found in isolation. These findings highlight the necessity for broad tumor mutational analysis, independent of traditional breast cancer subtype, to efficiently tailor personalized therapies in the clinic based on individual tumor genetic profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 962. doi:1538-7445.AM2012-962

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

A comprehensive profiling of colorectal cancer patients through multiplexed functional pathway signature and somatic mutation analysis for effective therapy selection.

e14103 Background: Colorectal cancer (CRC) is a leading cause of death in the developed nations. The development of effective forms of cytotoxic chemotherapy for colorectal cancer has been paralleled by the emergence of monoclonal antibodies against proteins that are thought to be important in the proliferation of malignant cells. Recent clinical studies showed that EGFR-targeting agent improved response rate when added to chemotherapy, however patients with KRAS mutations did not benefit from such treatment. Here we report a comprehensive profiling of pathwy signal proteins for their level of expression/activation and presence of somatic mutations in tumor tissues obtained from 136 CRC patients. Methods: The collaborative enzyme enhanced reactive immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Once target proteins are captured on the microarray-surface, channeling events between two detector enzymes in proximity enable the profiling of the target proteins with high sensitivity. In this study, tumor lysates were prepared from frozen tissues obtained from 136 CRC patients. In addition to a multiplexed analysis for panel of mutations in KRAS and BRAF genes, the level of expression and activation of HER1, HER2, HER3, cMET, cKIT, IGF-1R, PI3K, AKT, ERK and other pathway proteins were determined using CEER technology. Results: KRAS and BRAF mutations were found in ~40% (54/136) and ~5% (6/136) respectively in this cohort. The most frequent mutant alleles were G12D (14%) and G13D (13%). Varying levels of RTKs and associated activation patterns were observed. Levels of phosphorylated ERK and AKT correlated to expression/activation patterns of HER1, cMET, HER3 and prevalence of each biomarker along with pathway redundancy will be presented. Conclusions: A comprehensive profiling of multiplexed pathway/ somatic mutation analysis provides critical information needed to select the most effective targeted agents to overcome pathway redundancy /activating mutation related drug resistance through treatment combination or sequencing.

Superoxide Dismutase 2 (Sod2) is Overexpressed at an Early Stage During Colorectal Carcinogenesis: A Putative Target for Celecoxib Chemoprevention

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.

Circulating Free Tumor DNA in Patient Plasma is a Near-Perfect Marker for Metastatic Spread of Colorectal Cancer: A Study on 165 Patients Undergoing Surgical Treatment

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.

Abstract 4854: Mutations activating proliferation pathways in ovarian cancer

Abstract Ovarian malignancies may arise through different mechanisms of oncogenesis, which influence their treatment and are important for prognosis. Essential to carcinogenesis is the activation of proliferation pathways such as those directed by PI3 kinase or KRAS. New sequencing technologies may improve the detection of somatic mutations in tumor tissue, which is complicated by the low representation of a mutation within a tissue sample inevitably containing normal cells and subpopulations of malignant cells. We analyzed selected exons in PIK3CA, PIK3R, KRAS, BRAF, EGFR and ERBB2 in 34 primary malignant ovarian tumors, including 10 serous, 4 borderline, 11 endometrioid, and 9 mixed or rare subtypes. DNA was extracted from flash-frozen tumor tissues, amplified, and sequenced on a 454-GS-Flx Genome Sequencer. Expression of PTEN, p53, Bcl2, ALDH and Ki67 were assessed by immunohistochemistry on formalin-fixed tissues. Nine mutations were observed in PIK3CA, 5 in BRAF, 4 in KRAS, and 2 in EGFR. SNPs present in the amplicons analyzed allowed allelic imbalance to be estimated, showing possible amplification of EGFR in 9 of 20 informative samples, and of PIK3CA in 6 of 19 informative samples, none of which contained a point mutation. Endometrioid tumors frequently showed anomalies in PIK3CA, Bcl2 expression, and not p53 overexpression. P53 overexpression was typical of serous tumors, and was associated with the absence of mutations in any of the genes sequenced. Borderline and low grade tumors were characterized by BRAF mutation, no imbalance at EGFR, no anomalies at PIK3CA, and less frequent p53 overexpression. The absence of BRAF mutations in high grade tumors suggested that they do not evolve from borderline tumors. PIK3CA mutations were not mutually exclusive with KRAS mutations, confirming the non-redundancy of these intertwined signaling pathways. PTEN extinction was not significantly negatively associated with either point mutations or allelic imbalance at PIK3CA, consistent with different roles for PTEN loss and PI3-kinase activation at different stages of tumorogenesis, in spite of the close association of PTEN with the PI3-kinase pathway. No significant associations were observed between ALDH and Ki67 and any parameters measured. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2011-4854

Correlation of breast tumor pathogenesis and somatic mutations on the basis of mitochondrial DNA whole genome

To investigate the correlation of somatic mutations in whole genome of mitochondrial DNA (mtDNA) in patients with breast tumor. The DNA of tumor tissue and peripheral blood in 4 benign breast tumor patients from August 2009 to December 2009 in our hospital were extracted. The mtDNA whole genomes were amplified by polymerase chain reaction (PCR) and the mutations of products screened by sequencing to compare the difference of mutation distribution between tumor tissue and peripheral blood. The likelihood of somatic mutations in tumor tissue was determined. The mutation spectrum of mtDNA genomes was obtained by PCR and sequencing. Then a phylogenetic tree was constructed to identify their haplogroups (D4i, G1, R9b, N9a). Their private mutations in peripheral blood were detected and the somatic mutation at 16292 position was found in one patient (haplogroups: R9b). Based on the extensively study on the mtDNA genomes from the tumor issues of 4 patients, our current report observed only a single somatic variation from the control region, no any further mutations with potential function from the coding region were observed.

Relationship between K-ras oncogene mutations in portal vein blood of patients with colorectal cancer and liver metastases

Objective To detect mutations of K-ras oncogene in portal vein blood of patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer. Methods Forty-eight patients with colorectal cancer were screened for the mutations of K-ras oncogene in tissue samples from their tumors, portal vein blood, proximally adjacent mucosa and 8metastatic liver biopsies by real-time fluorescence quantitative polymerase chain reaction (PCR) and DNA sequencing. The results were analyzed with their clinical data. Results Sixteen of the 48 patients with colorectal cancer had K-ras point mutations at codon 12 in their portal vein blood, and 17 of 48 patients had K-ras mutations in their primary tumors, but only 4 of 48 patients had K-ras mutations in proximally adjacent mucosa. There was no significant difference in rate of K-ras mutation between tumor tissues and portal vein blood (P ＞ 0. 05 ), but significant difference was found between the tumor tissue and the proximally adjacent mucosa ( P ＜0. 05 ). The rate of K-ras mutations in portal vein blood of colorectal cancer with liver metastases (70. 0% ) was higher than that of without liver metastases (23.7%). Sixteen cases of mutated K-ras in portal vein blood showed mutations in tumor tissues. Patients without mutated K-ras in tumor tissue had no mutations in their portal vein blood and proximally adjacent mucosa. In 5 of 8 patients with simultaneous liver metastasis, mutated K-ras oncogenes were detected in portal vein blood, and the type of K-ras mutation detected in the tumor tissue was accord with that in metastatic liver biopsies. Two patients with mutated K-ras detected in their portal vein blood had no liver metastases during perioperation, but liver metastases were diagnosed by CT at the postoperative month 6 and 9 respectively. The main types of K-ras mutations at codon 12 included GGT to GAT and GGT to GTT. No one had point mutation at codon 13. Conclusion Mutated K-ras detected in both cancer tissue and portal vein blood may indicate livermetastases from colorectal cancer. Key words: Colorectal neoplasms; Ras gene; Mutation; Hepatic metastasis

Phase II Study of Dasatinib in Patients With Advanced Non–Small-Cell Lung Cancer

Src family kinases (SFKs) promote cancer progression and are commonly expressed in non-small-cell lung cancer (NSCLC), but the clinical effects of SFK inhibition in NSCLC are unknown. We conducted a phase II trial of the SFK inhibitor dasatinib for advanced NSCLC. We tested the hypotheses that the activation of epidermal growth factor receptor (EGFR) or SFK or modulation of serum cytokines may predict a response to dasatinib. Patients received dasatinib as first-line therapy. Response was measured by tumor size on computed tomography scans and by metabolic activity on positron emission tomography scans. Tissue samples taken before patients received dasatinib were tested for EGFR and Kras mutation and phosphorylated SFK expression. Thirty-four patients were enrolled. The overall disease control rate (partial responses plus stable disease) for dasatinib was 43%. One patient had a partial response to therapy. Eleven patients (32%) had a metabolic response to dasatinib. SFK activation and EGFR and Kras mutations in tumor tissue did not predict response to dasatinib. Significant toxicities included fatigue and dyspnea. The presence of a pleural effusion before dasatanib therapy predicted the development of a clinically significant effusion during therapy. Dasatinib as a single agent had modest clinical activity that was lower than that generally observed in patients with NSCLC who receive chemotherapy. Pleural effusion was an expected and problematic toxicity that was successfully treated with steroids, diuretics, and dose interruptions. Marked activity in one patient and prolonged stable disease in four others suggested a potential subpopulation of patients with dasatinib-sensitive NSCLC.

Detection of phosphoinositide-3-kinase, catalytic, and alpha polypeptide (PIK3CA) mutations in matched tissue and plasma samples from patients with metastatic breast cancer.

10502 Background: The PIK3CA gene is mutated in ∼25% of breast cancer cases and has been proposed as a potential biomarker for targeted therapies. The analysis of PIK3CA mutations in tumor tissue could therefore be an important step in the treatment decisions process. In cases where tumor tissue is not available, the detection of PIK3CA mutations in circulating plasma DNA might provide a noninvasive alternative. In order to determine the feasibility of plasma-based mutation testing, we analyzed the PIK3CA mutation status in matched tissue and plasma samples from patients with metastatic breast cancer. Methods: We used a highly sensitive and quantitative digital PCR-based approach, named BEAMing, to detect and quantify the four most common PIK3CA mutations (E542K, E545K, H1047R, H1047L) in exon 9 and 20, representing ∼87% of all reported PIK3CA mutations. We included 50 cases with metastatic breast cancer (stage IV) and assessed the mutational status of DNA extracted from FFPE tissue of the primary tumor and matched plasma. Results: PIK3CA mutations were detected in 15 out of 50 tissue sections (30%). The fraction of mutant to wild-type DNA molecules (mutational load) in the tissue samples ranged from 3.9% to 44%, with a median fraction of 15%. Identical mutations could be detected in all matched plasma samples. Mutational load in the plasma samples ranged from 0.061% to 22% in the 15 PIK3CA mutant plasma samples (median: 6.6%), representing 2 to 9,906 mutant molecules per ml plasma (median: 370 molecules). None of the patients with a PIK3CA negative tumor tissue section revealed PIK3CA mutations in the plasma DNA resulting in a 100% concordance between tissue and plasma samples. Conclusions: The detection of PIK3CA mutations in plasma DNA by BEAMing can be used as an alternative approach to tissue analysis in metastatic breast cancer patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Indivumed, Inostics Indivumed, Inostics

Comparison of different methods for detecting epidermal growth factor receptor mutations in peripheral blood as a predictor of response to gefitinib.

10557 Background: Several studies have reported the detection of active EGFR mutation DNA in peripheral blood can also predict the response to EGFR-TKI in NSCLC. But heterogeneity in methodologies makes it difficult to use in daily practice. Goal of this study is to compare different methods for detecting EGFR mutation DNA in peripheral blood. Methods: From July 2007 to April 2009, fifty-one advanced NSCLC patients with pairs of tumour and serum samples treated with gefitinib were included. Response was evaluated by RECIST criteria. EGFR mutations in tumor tissues were detected by Scorpions-Amplification Refractory Mutation System (Scorpions-ARMS). Three methods were used to detect the mutations DNA in peripheral blood: denaturing high performance liquid chromatography (DHPLC), Mutant-Enriched-Liquidchip (ME-Liquidchip) and Scorpions-ARMS. Results: The Objective response rates (ORR) in whole group patients are 33.3% (17/51). EGFR Mutation rate were identified 15.7% (8/51), 27.5% (14/51), 29.4% (15/51) by DHPLC, ME-Liquidchip, Scorpions-ARMS in peripheral blood respectively. Only 34 cases were available for paired tumor sample EGFR mutation detection. In these 34 patients, EGFR mutation rate in tumor tissue was 41.2% (14/34). The corresponding rates of EGFR mutation status in peripheral blood with tumor tissues are 58.8% (by DHPLC), 58.8% (by ME-Liquidchip) and 70.6% (by Scorpions-ARMS). The ORRs by different method of EGFR mutation detection were 71.4% (Tumor sample), 62.5% (Blood DHPLC), 50.0% (Blood, ME-Liquidchip) and 66.7% (Blood, Scorpions-ARMS). EGFR Mutation detected by Scorpions-ARMS in tumor tissues and peripheral blood have a better prediction of ORR of EGFR-TKI (p=0.002 and p=0.003) compare DHPLC and ME-Liquidchip method. Conclusions: Tumor tissue is the better source of samples to detect EGFR mutation of NSCLC patients than peripheral blood. Scorpions-ARMS method is superior to DHPLC and ME-Liquidchip in serum EGFR mutation DNA detection. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Innovation Centre China, R&D AstraZeneca, Surexam, Inc AstraZeneca, GlaxoSmithKline AstraZeneca, Bayer, Novartis, sanofi-aventis

Low correspondence of EGFR mutations in tumor tissue and paired serum of non-small-cell lung cancer patients

Objective: Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumor response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC) patients. The aim of this study was to evaluate the correspondence between EGFR mutations in non-small-cell lung cancer tissues and in circulating DNA. Methods: The research was conducted in 50 non-small-cell lung cancer patients who had undergone curative surgery, and in whom both serum and neoplastic tissues were available. Meanwhile sera of 33 cases of advanced NSCLC patients were also analyzed. DNA were extracted from each sample. Mutations of EGFR in exon18-21 were examined by PCR amplification method and direct sequencing. Results: EGFR mutations were detected in 15 (30%) of 50 neoplastic tissue samples, 6 cases were in-frame deletion del E746-A750 in exon19, 9 cases were substitution in exon 21 (all were L858R except one was L861Q), but no mutated DNA resulted in paired serum circulating DNA samples of 50 resectable patients. As the 33 advanced NSCLC patients, EGFR mutations were detected in only 2 serum circulating DNA samples, all were L858R mutation in exon 21. Conclusion: These data indicated that it was difficult to identify EGFR mutations in circulating DNA of NSCLC patients. The use of EGFR mutation in serum as a clinical method for decision making of TKI therapy is unsatisfactory.

Lung cancer susceptibility

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