[Show abstract][Hide abstract] ABSTRACT: Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth. Variant calling using an unmatched Hapmap DNA control removed a substantial number of artifactual calls regardless of algorithm used or variant class. The removed calls were nonunique, had lower variant frequencies, and tended to recur in multiple unrelated samples. Analysis of sample replicates revealed that reproducible calls had distinctly higher variant allele depths and frequencies compared to nonreproducible calls. On the basis of these differences, filters on variant frequency were chosen to select for reproducible calls. The analysis pipeline successfully retrieved the associated known variant in all tested samples and uncovered additional mutations in some samples corresponding to well-characterized hotspot mutations in acute myeloid leukemia. We have developed a paired-sample analysis pipeline capable of robust identification of mutations from microdroplet-PCR sequencing data with high sensitivity and specificity.
[Show abstract][Hide abstract] ABSTRACT: More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.
Genes & development 09/2013; 27(18):1986-98. · 12.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most gastrointestinal stromal tumors (GISTs) can be recognized by their monotonous cytologic features and overexpression of KIT oncoprotein. Altered morphology and loss of CD117 reactivity has been described previously after chronic imatinib treatment; however, this phenomenon has not been reported in imatinib-naive tumors. Eight patients with abrupt transition from a classic CD117-positive spindle cell GIST to an anaplastic CD117-negative tumor were investigated for underlying molecular mechanisms of tumor progression. Pathologic and molecular analysis was performed on each of the 2 components. Genomic DNA polymerase chain reaction for KIT, PDGFRA, BRAF, and KRAS hot spot mutations and fluorescence in situ hybridization for detecting KIT gene copy number alterations were performed. TP53 mutational analysis was performed in 5 cases. There were 7 men and 1 woman, with an age range of 23 to 65 years. Five of the primary tumors were located in the stomach, and 1 case each originated in the small bowel, colon, and rectum. In 3 patients, the dedifferentiated component occurred in the setting of imatinib resistance, whereas the remaining 5 occurred de novo. The dedifferentiated component had an anaplastic appearance, including 1 angiosarcomatous phenotype, with high mitotic activity and necrosis, and showed complete loss of CD117 (8/8) and CD34 (5/8) expression and de novo expression of either cytokeratin (4/8) or desmin (1/8). There was no difference in the KIT genotype between the 2 components. However, 2 imatinib-resistant tumors showed coexistence of KIT exon 11 and exon 13 mutations. Fluorescence in situ hybridization showed loss of 1 KIT gene in 3 cases and low-level amplification of KIT in 2 other cases in the CD117-negative component, compared with the CD117-positive area. TP53 mutation was identified in 1/5 cases tested, being present in both components. In summary, dedifferentiation in GIST may occur either de novo or after chronic imatinib exposure and can represent a diagnostic pitfall. This phenomenon is not related to additional KIT mutations, but might be secondary to genetic instability, either represented by loss of heterozygosity or low level of KIT amplification.
The American journal of surgical pathology 01/2013; · 4.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) genotyping is now standard in the management of advanced lung adenocarcinoma, as this biomarker predicts marked benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). EGFR exon 19 insertions are a poorly described family of EGFR mutations, and their association with EGFR-TKI sensitivity in lung adenocarcinoma is uncertain.
Patients with lung cancers harboring EGFR exon 19 insertions were studied. The predicted effects of the insertions on the structure of the EGFR protein were examined, and EGFR exon 19 insertions were introduced into Ba/F3 cells to assess oncogenicity and in vitro sensitivity to EGFR-TKIs. In patients receiving TKI, response magnitude was assessed with serial computed tomographic (CT) measurement.
Twelve tumors harboring EGFR exon 19 insertions were identified; patients were predominately female (92%) and never-smokers (75%). The 11 specimens available for full sequencing all showed an 18-bp insertion that resulted in the substitution of a Pro for Leu at residue 747. The mutant EGFR transformed the Ba/F3 cells, which were then sensitive to EGFR-TKI. Six patients with measurable disease received TKI and five had a response on serial CT.
EGFR exon 19 insertions are a newly appreciated family of EGFR-TKI-sensitizing mutations, and patients with tumors harboring these mutations should be treated with EGFR-TKI. While these mutations may be missed through the use of some mutation-specific assays, the addition of PCR product size analysis to multigene assays allows sensitive detection of both exon 19 insertion and deletion mutations.
Clinical Cancer Research 12/2011; 18(6):1790-7. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT).
We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing.
Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively.
In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies.
Clinical Cancer Research 12/2011; 18(3):748-57. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The epidermal growth factor receptor (EGFR) mutation T790M is reported in approximately 50% of lung cancers with acquired resistance to EGFR inhibitors and is a potential prognostic and predictive biomarker. Its assessment can be challenging due to limited tissue availability and underdetection at low mutant allele levels. Here, we sought to determine the feasibility of tumor rebiopsy and to more accurately assess the prevalence of the T790M using a highly sensitive locked nucleic acid (LNA) PCR/sequencing assay. MET amplification was also analyzed. Methods: Patients with acquired resistance were rebiopsied and samples were studied for sensitizing EGFR mutations. Positive cases were evaluated for T790M using standard PCR-based methods and a subset were re-evaluated with an LNA-PCR/sequencing method with an analytical sensitivity of approximately 0.1%. MET amplification was assessed by FISH. RESULTS: Of 121 patients undergoing tissue sampling, 104 (86%) were successfully analyzed for sensitizing EGFR mutations. Most failures were related to low tumor content. All patients (61/61) with matched pretreatment and resistance specimens showed concordance for the original sensitizing EGFR mutation. Standard T790M mutation analysis on 99 patients detected 51(51%) mutants. Retesting of 30 negative patients by the LNA-based method detected 11 additional mutants for an estimated prevalence of 68%. MET was amplified in 11% of cases (4/37). CONCLUSIONS: The re-biopsy of lung cancer patients with acquired resistance is feasible and provides sufficient material for mutation analysis in most patients. Using high sensitivity methods, the T790M is detected in up to 68% of these patients.
Clinical Cancer Research 01/2011; 17(5):1169-80. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: The BRAF V600E mutation has been associated with aggressive disease in papillary thyroid carcinoma (PTC). Molecular testing has been proposed as a useful adjunct to cytology in the diagnosis of malignancy and for tailoring clinical management. The aims of our study were to evaluate the BRAF mutational status using archived fine-needle aspiration biopsy (FNAB) material from patients with long-term follow-up and to correlate it with the original cytology diagnosis, clinicopathological stage at surgery, and prognosis. Study Design: FNAB material from 52 cases of PTC, with a mean follow-up of 8.4 years, was used in this study. DNA was extracted from archival cytology slides. Mutation analysis was performed by standard sequencing and locked nucleic acid-PCR/sequencing. Results: The BRAF V600E mutation was present in 46% of cases, but it was absent in all FNABs diagnosed originally as atypical and in 14 of 17 suspicious cases. Recurrence was significantly more frequent (p = 0.006) in cases with BRAF mutations and 54% of these cases presented with stage 2 or higher. Conclusion: The BRAF V600E mutation is associated with a higher pathological stage at surgery and a higher rate of recurrence. BRAF mutation analysis did not provide a significant increase in the accuracy of thyroid FNABs diagnosed as suspicious or atypical in our institution.
[Show abstract][Hide abstract] ABSTRACT: KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening.
The Journal of molecular diagnostics: JMD 01/2011; 13(1):64-73. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Germ line polymorphisms may confer susceptibility to lung cancer in never smokers, but studies in the United States have been limited by the low number of cases seen at single institutions. We hypothesized that we could use the Internet to bolster the accrual of appropriate patients.
We established an Internet-based protocol to collect blood and information from patients throughout the United States. To illustrate the power of this approach, we used these samples, plus additional cases and age-matched controls from the Memorial Sloan-Kettering Cancer Center (New York, NY) and the Aichi Cancer Center (Nagoya, Japan), to analyze germ line DNA for genetic variants reportedly associated with lung cancer susceptibility. The genotypes for the polymorphisms rs763317 (intron 1) and T790M (exon 20) in the EGFR gene were determined by direct sequencing, and CHRNA3 nicotinic acetylcholine receptor single nucleotide polymorphisms (rs8034191 and rs1051730) were genotyped as part of a pilot genome-wide association study.
We successfully analyzed germ line DNA from 369 cases, including 45 obtained via the Internet, and 342 controls. A germ line EGFR T790M variant was identified in 2 of the 369 cases (0.54%; 95% confidence interval, 0.21-1.29%), and in none of the 292 controls (P = 0.21). No difference was observed in EGFR rs763317 frequency between cases and controls. Similarly, neither CHRNA3 rs8034191 nor rs1051730 were associated with lung cancer risk.
The Internet provides a way to recruit patients throughout the country for minimal risk studies. This approach could be used to facilitate studies of germ line polymorphisms in specific groups of patients with cancer.
Clinical Cancer Research 01/2010; 16(2):755-63. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics.
The Journal of molecular diagnostics: JMD 08/2009; 11(4):266-78. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and the anti-EGFR monoclonal antibodies have proven activity in lung and colorectal adenocarcinomas, respectively, but only a small fraction of patients exhibit significant responses. The observation that only a minority of patients respond to EGFR-targeted therapies, in combination with their toxicity and high costs, has driven the search for molecular markers predictive of response. The main focus of the present review is the recent discovery that mutations in the KRAS oncogene constitute a negative predictive marker in this clinical setting, namely that their presence can be used to predict which patients are unlikely to benefit from treatment with EGFR-directed therapy.
Seminars in Diagnostic Pathology 12/2008; 25(4):288-94. · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare non-truncating sequence variants in the BRCA1 and BRCA2 genes is problematic because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Several studies have reported the biochemical analysis of BRCA2 variants, which disrupted the 5' and 3'splicing consensus elements (the GU-AG rule). However, little has been done to look into the consequences of variants located outside the 5' and 3' consensus splice sites. cDNA analysis demonstrates that the BRCA2*IVS4-12del5 splice site variant results in the deletion of exon 5, and the gene putatively produces a truncated BRCA2 protein of 164 amino acids instead of 3418 with the incorporation of 22 out of frame amino acids. The pattern of breast, melanoma, and pancreatic cancers in the paternal kindred is consistent with autosomal dominant inheritance of a deleterious BRCA2 mutation. Analysis of a tumor specimen indicates loss of heterozygosity (LOH). Sequence alignment indicates the deleted region is well conserved across different species. These results support the conclusion that BRCA2 IVS4-12del5 is a deleterious mutation. This study will shed light on the reclassification of intronic variants that do not disrupt the 5' and 3' splice sites (the GU-AG rule).
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2008; 663(1-2):84-9. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KRAS mutations are found in approximately 25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib.
We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas.
KRAS mutational analysis was done on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12 of 81; 95% confidence intervals, 8-24%) of tumors from never smokers. Similarly, 22% (69 of 316; 95% confidence intervals, 17-27%) of tumors from former smokers, and 25% (21 of 85; 95% confidence intervals, 16-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G-->A) rather than the transversion mutations known to be smoking-related (G-->T or G-->C; P < 0.0001).
Based on our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.
Clinical Cancer Research 10/2008; 14(18):5731-4. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular hallmark of angiomatoid fibrous histiocytoma (AFH) is not well defined, with only six cases with specific gene fusions reported to date, consisting of either FUS-ATF1 or EWSR1-ATF1. To address this, we investigated the presence of FUS-ATF1, EWSR1-ATF1, and the highly related EWSR1-CREB1 fusion in a group of nine AFHs. All cases were subjected to RT-PCR for EWSR1-ATF1 and EWSR1-CREB1. FISH for EWSR1 and FUS rearrangements was performed in most cases. Transcriptional profiling was performed in three tumors and their gene expression was compared to five clear cell sarcomas expressing either the EWSR1-ATF1 or EWSR1-CREB1 fusion. By RT-PCR, eight out of nine tumors showed the presence of the EWSR1-CREB1 fusion, while one had an EWSR1-ATF1 transcript. FISH showed evidence of EWSR1 rearrangement in seven out of eight cases. Karyotypic analysis performed in one tumor showed a t(2;22)(q33;q12). High transcript levels were noted for TFE3 in AFH tumors, while overexpression of genes involved in melanogenesis, such as MITF, GP100, and MET was noted in somatic clear cell sarcomas. We report for the first time the presence of EWSR1-CREB1 in AFH, which now appears to be the most frequent gene fusion in this tumor. EWSR1-CREB1 is a novel translocation recently described in clear cell sarcoma of the GI tract. EWSR1-ATF1, identified in some AFH cases, is the most common genetic abnormality in soft tissue clear cell sarcoma. Thus, identical fusions involving ATF1 and CREB1 are found in two distinct sarcomas, which may be able to transform two different types of mesenchymal precursor cells, unlike most other sarcoma gene fusions.
Genes Chromosomes and Cancer 01/2008; 46(12):1051-60. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study summarizes our initial experience with prospective, single-amplicon (mutation-specific) A636P testing in Ashkenazi Jewish patients at risk for Hereditary Nonpolyposis Colorectal Cancer (HNPCC).
We previously described a founder mutation, MSH2*1906G >C (A636P) that causes HNPCC in 8/1345 (0.59%) of Ashkenazim with colorectal cancer. The mutation was more common in Ashkenazim diagnosed at <or=40 years (7%).
Twenty-seven Ashkenazi probands at risk for HNPCC were ascertained. Single-amplicon A636P testing was performed on 21 by polymerase chain reaction of exon 12 of MSH2, followed by direct DNA sequencing. Mutational analysis of the entire open reading frame of MLH1 and MSH2 was performed on 7 by PCR of each exon, followed by heteroduplex analysis using denaturing high-performance liquid chromatography and direct sequencing of exons with variant chromatographs. One patient received both studies,
The A636P mutation was detected in 3/21 (14%) prospectively evaluated patients using single amplicon testing. In 6 patients, the entire open reading frame of MLH1 and MSH2 was analyzed, and 1 additional A636P carrier and 2 carriers of previously unrecognized mutations were identified. The A636P mutation was present in 2 patients who met Amsterdam criteria and in 2 patients who did not.
Although rare in the general population, A636P mutations are found at increased frequency in Ashkenazim with a personal or family history of colorectal or other HNPCC-associated cancers. This inexpensive and rapid approach may be useful preoperatively in helping determine the extent of colon resection for a subset of patients.
Annals of Surgery 04/2007; 245(4):560-5. · 6.33 Impact Factor