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1.  Dedifferentiation in Gastrointestinal Stromal Tumor to an Anaplastic KIT Negative Phenotype – a Diagnostic Pitfall. Morphologic and Molecular Characterization of 8 Cases Occurring either de-novo or after Imatinib Therapy 
Most 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-naïve 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 two components. Genomic DNA PCR for KIT, PDGFRA, BRAF and KRAS hot spot-mutations and FISH for detecting KIT gene copy number alterations were performed. TP53 mutational analysis was performed in 5 cases. There were 7 males and 1 female, with an age range of 23–65 years. Five of the primary tumors were located in the stomach, while one case each originated in small bowel, colon and rectum. In 3 patients, the dedifferentiated component occurred in the setting of imatinib-resistance, while the remaining 5 occurred de novo. The dedifferentiated component had an anaplastic appearance, including one 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 two components. However, two imatinib-resistant tumors showed co-existence of KIT exon 11 and exon 13 mutations. FISH showed loss of one KIT gene in 3 cases and low level amplification of KIT in 2 other cases in the CD117-negative component, compared to 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.
PMCID: PMC3728887  PMID: 23348204
gastrointestinal stromal tumor; dedifferentiation; KIT; imatinib
2.  EGFR Exon 20 Insertion Mutations in Lung Adenocarcinomas: Prevalence, Molecular Heterogeneity, and Clinicopathologic Characteristics 
Molecular cancer therapeutics  2013;12(2):220-229.
In contrast to other primary EGFR mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1 and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases (2.2%, 95% CI 1.6 to 3.1%), all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (p<0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations, but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12bp, resulting in 13 different insertions which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors.
PMCID: PMC3714231  PMID: 23371856
EGFR exon 20; EGFR; epidermal growth factor receptor; lung adenocarcinoma; driver oncogenes
3.  Prevalence, clinicopathologic associations and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas 
Activating mutations in the tyrosine kinase domain of HER2 (ERBB2) have been described in a subset of lung adenocarcinomas (ADCs) and are mutually exclusive with EGFR and KRAS mutations. The prevalence, clinicopathologic characteristics, prognostic implications, and molecular heterogeneity of HER2-mutated lung ADCs are not well established in US patients.
Experimental Design
Lung ADC samples (n=1478) were first screened for mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and negative cases were then assessed for HER2 mutations (exons 19–20) using a sizing assay and mass spectrometry. Testing for additional recurrent point mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, MEK1 and AKT was performed by mass spectrometry. ALK rearrangements and HER2 amplification were assessed by FISH.
We identified 25 cases with HER2 mutations, representing 6% of EGFR/KRAS/ALK-negative specimens. Small insertions in exon 20 accounted for 96% (24/25) of the cases. Compared to insertions in EGFR exon 20, there was less variability, with 83% (20/24) being a 12bp insertion causing duplication of amino acids YVMA at codon 775. Morphologically, 92% (23/25) were moderately or poorly differentiated ADC. HER2 mutation was not associated with concurrent HER2 amplification in 11 cases tested for both. HER2 mutations were more frequent among never-smokers (p<0.0001) but there were no associations with sex, race, or stage.
HER2 mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer worldwide and the availability of standard and investigational therapies targeting HER2, routine clinical genotyping of lung ADC should include HER2.
PMCID: PMC3865806  PMID: 22761469
HER2; ERBB2; lung adenocarcinoma; EGFR; driver oncogenes
4.  EGFR Exon 19 Insertions: A New Family of Sensitizing EGFR Mutations in Lung Adenocarcinoma 
Clinical Cancer Research  2011;18(6):1790-1797.
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 (TKIs). EGFR exon 19 insertions are a poorly described family of EGFR mutations, and their association with EGFR TKI-sensitivity in lung adenocarcinoma is uncertain.
Experimental Design
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 tomography (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 demonstrated 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 5 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 multi-gene assays allows sensitive detection of both exon 19 insertion and deletion mutations.
PMCID: PMC3306520  PMID: 22190593
Clinical Cancer Research  2011;18(3):748-757.
Compared to the numerous broad screens for oncogene mutations in adult cancers, very few have been performed in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we performed a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma (NB), Ewing sarcoma (ES), rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT).
Experimental Design
We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 NB, 75 ES, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing.
Mutations were identified in 13% of NB samples, 4% of ES samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of NB samples. The remainder of NB 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 (ARMS) (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (Beta-Catenin) mutations in 4.9% and 3.3% of ERMS, respectively.
In ERMS, ES, and NB, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, NB and ERMS contain significant subsets of cases with non-overlapping 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.
PMCID: PMC3271129  PMID: 22142829
mutation; rhabdomyosarcoma; neuroblastoma; Ewing sarcoma; desmoplastic small round cell tumor
6.  Rebiopsy of Lung Cancer Patients with Acquired Resistance to EGFR Inhibitors and Enhanced Detection of the T790M Mutation Using a Locked Nucleic Acid-Based Assay 
The 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 is also analyzed.
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.
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).
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.
PMCID: PMC3070951  PMID: 21248300
8.  Analysis of genetic variants in never-smokers with lung cancer facilitated by an Internet-based blood collection protocol: a preliminary report 
Germline polymorphisms may confer susceptibility to lung cancer in never smokers, but studies in the US have been limited by the low number of cases seen at single institutions. We hypothesized that we could use the Internet to bolster accrual of appropriate patients.
Experimental Design
We established an Internet-based protocol to collect blood and information from patients throughout the U.S. To illustrate the power of this approach, we used these samples, plus additional cases and age-matched controls from Memorial Sloan-Kettering Cancer Center (New York, NY) and the Aichi Cancer Center (Nagoya, Japan), to analyze germline 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 SNPs (rs8034191 and rs1051730) were genotyped as part of a pilot genome-wide association study.
We successfully analyzed germline DNA from 369 cases, including 45 obtained via the Internet, and 342 controls. A germline EGFR T790M variant was identified in 2 (0.54%, 95% CI: 0.21%–1.29%) of the 369 cases, and in none of the 292 controls (p=0.208). No difference was observed in EGFR rs763317 frequency between cases and controls. Similarly, neither CHRNA3 rs8034191 nor rs1051730 was 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 germline polymorphisms in specific groups of patients with cancer.
PMCID: PMC2808124  PMID: 20068085
Non-small cell lung cancer; EGFR; never smoker; genetic susceptibility; Internet
9.  Frequency and Distinctive Spectrum of KRAS Mutations in Never Smokers with Lung Adenocarcinoma 
KRAS mutations are found in ~ 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.
Experimental Design:
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 performed on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12/81; 95% CI 8%-24%) of tumors from never smokers. Similarly, 22% (69/316; 95% CI 17%-27%) of tumors from former smokers, and 25% (21/85; 95% CI 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 upon 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.
PMCID: PMC2754127  PMID: 18794081
10.  Prediction of Germline Mutations and Cancer Risk in the Lynch Syndrome 
Identifying families at high risk for the Lynch syndrome (ie, hereditary non-polyposis colorectal cancer) is critical for both genetic counseling and cancer prevention. Current clinical guidelines are effective but limited by applicability and cost.
To develop and validate a genetic counseling and risk prediction tool that estimates the probability of carrying a deleterious mutation in mismatch repair genes MLH1, MSH2, or MSH6 and the probability of developing colorectal or endometrial cancer.
Design, Setting, and Patients
External validation of the MMRpro model was conducted on 279 individuals from 226 clinic-based families in the United States, Canada, and Australia (referred between 1993–2005) by comparing model predictions with results of highly sensitive germline mutation detection techniques. MMRpro models the autosomal dominant inheritance of mismatch repair mutations, with parameters based on meta-analyses of the penetrance and prevalence of mutations and of the predictive values of tumor characteristics. The model’s prediction is tailored to each individual’s detailed family history information on colorectal and endometrial cancer and to tumor characteristics including microsatellite instability.
Main Outcome Measure
Ability of MMRpro to correctly predict mutation carrier status, as measured by operating characteristics, calibration, and overall accuracy.
In the independent validation, MMRpro provided a concordance index of 0.83 (95% confidence interval, 0.78–0.88) and a ratio of observed to predicted cases of 0.94 (95% confidence interval, 0.84–1.05). This results in higher accuracy than existing alternatives and current clinical guidelines.
MMRpro is a broadly applicable, accurate prediction model that can contribute to current screening and genetic counseling practices in a high-risk population. It is more sensitive and more specific than existing clinical guidelines for identifying individuals who may benefit from MMR germline testing. It is applicable to individuals for whom tumor samples are not available and to individuals in whom germline testing finds no mutation.
PMCID: PMC2538673  PMID: 17003396
11.  Frequency of CHEK2*1100delC in New York breast cancer cases and controls 
The 1100delC CHEK2 allele has been associated with a 1.4–4.7 fold increased risk for breast cancer in women carrying this mutation. While the frequency of 1100delC was 1.1–1.4% in healthy Finnish controls, the frequency of this allele in a North American control population and in North American breast cancer kindreds remains unclear.
We genotyped 1665 healthy New York volunteers and 300 cases of breast cancer for the CHEK2*1100delC.
The overall frequency of the 1100delC was 3/300 (1.0%) among all cases with either a family history of breast cancer (n = 192) or a personal history of breast cancer (n = 108, of which 46 were bilateral, 46 unilateral, and 16 were male breast cancer cases), compared to a frequency of 5/1665 (0.3%) in healthy controls (p = 0.1). There was no difference in allele frequency among Ashkenazi and non-Ashkenazi controls.
The relatively low breast cancer penetrance of this allele, along with the low population frequency, will limit the clinical applicability of germline testing for CHEK2*1100delC in North American kindreds.
PMCID: PMC149355  PMID: 12529183

Results 1-11 (11)