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1.  Patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias 
Leukemia  2016;31(1):151-158.
Genomic studies have identified recurrent somatic mutations in acute leukemias. However, current murine models do not sufficiently encompass the genomic complexity of human leukemias. To develop pre-clinical models, we transplanted 160 samples from patients with acute leukemia (AML, MLL, B-ALL and T-ALL) into immunodeficient mice. Of these, 119 engrafted with expected immunophenotype. Targeted sequencing of 374 genes and 265 frequently rearranged RNAs detected recurrent and novel genetic lesions in 48 paired primary tumor (PT) and patient-derived xenotransplant (PDX) samples. Overall, the frequencies of 274 somatic variant alleles correlated between PT and PDX samples, although the data were highly variable for variant alleles present at 0-10%. 17% of variant alleles were detected in either PT or PDX samples only. Based on variant allele frequency changes, 24 PT-PDX pairs were classified as concordant while the other 24 pairs showed various degree of clonal discordance. There was no correlation of clonal concordance with clinical parameters of diseases. Significantly more bone marrow samples than peripheral blood samples engrafted discordantly. These data demonstrate the utility of developing PDX banks for modeling human leukemia, and emphasize the importance of genomic profiling of PDX and patient samples to ensure concordance before performing mechanistic or therapeutic studies.
PMCID: PMC5203983  PMID: 27363283
2.  Comprehensive Genomic Profiling of Advanced Penile Carcinoma Suggests a High Frequency of Clinically Relevant Genomic Alterations 
The Oncologist  2015;21(1):33-39.
Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs). CGP identified CRGAs in patients with advanced penile squamous cell carcinoma, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients.
Advanced penile squamous cell carcinoma (PSCC) is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs).
Materials and Methods.
DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded sections in patients with advanced PSCC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 692× for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. CRGAs were defined as genomic alterations (GAs) linked to targeted therapies on the market or under evaluation in mechanism-driven clinical trials.
Twenty male patients with a median age of 60 years (range, 46–87 years) were assessed. Seventeen (85%) cases were stage IV and three cases (15%) were stage III. CGP revealed 109 GAs (5.45 per tumor), 44 of which were CRGAs (2.2 per tumor). At least one CRGA was detected in 19 (95%) cases, and the most common CRGAs were CDKN2A point mutations and homozygous deletion (40%), NOTCH1 point mutations and rearrangements (25%), PIK3CA point mutations and amplification (25%), EGFR amplification (20%), CCND1 amplification (20%), BRCA2 insertions/deletions (10%), RICTOR amplifications (10%), and FBXW7 point mutations (10%).
CGP identified CRGAs in patients with advanced PSCC, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients.
Implications for Practice:
Few treatment options exist for patients with advanced penile squamous cell carcinoma (PSCC). Outcomes are dismal with platinum-based chemotherapy, with median survival estimated at 1 year or less across multiple series. Biological studies of patients with PSCC to date have principally focused on human papillomavirus status, but few studies have elucidated molecular drivers of the disease. To this end, comprehensive genomic profiling was performed in a cohort of 20 patients with advanced PSCC. Findings of frequent mutations in CDKN2A, NOTCH1, PIK3CA, and EGFR (all in excess of 20%) point to potential therapeutic avenues. Trials of targeted therapies directed toward these mutations should be explored.
PMCID: PMC4709208  PMID: 26670666
Penile cancer; Sequencing; Targeted therapy; Mutation; Genomic profiling; Human papillomavirus
3.  Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome 
PLoS Genetics  2016;12(12):e1006501.
Neuroblastoma is characterized by a relative paucity of recurrent somatic mutations at diagnosis. However, recent studies have shown that the mutational burden increases at relapse, likely as a result of clonal evolution of mutation-carrying cells during primary treatment. To inform the development of personalized therapies, we sought to further define the frequency of potentially actionable mutations in neuroblastoma, both at diagnosis and after chemotherapy. We performed a retrospective study to determine mutation frequency, the only inclusion criterion being availability of cancer gene panel sequencing data from Foundation Medicine. We analyzed 151 neuroblastoma tumor samples: 44 obtained at diagnosis, 42 at second look surgery or biopsy for stable disease after chemotherapy, and 59 at relapse (6 were obtained at unknown time points). Nine patients had multiple tumor biopsies. ALK was the most commonly mutated gene in this cohort, and we observed a higher frequency of suspected oncogenic ALK mutations in relapsed disease than at diagnosis. Patients with relapsed disease had, on average, a greater number of mutations reported to be recurrent in cancer, and a greater number of mutations in genes that are potentially targetable with available therapeutics. We also observed an enrichment of reported recurrent RAS/MAPK pathway mutations in tumors obtained after chemotherapy. Our data support recent evidence suggesting that neuroblastomas undergo substantial mutational evolution during therapy, and that relapsed disease is more likely to be driven by a targetable oncogenic pathway, highlighting that it is critical to base treatment decisions on the molecular profile of the tumor at the time of treatment. However, it will be necessary to conduct prospective clinical trials that match sequencing results to targeted therapeutic intervention to determine if cancer genomic profiling improves patient outcomes.
Author Summary
Neuroblastoma is a pediatric cancer that usually affects children within the first five years of life. The survival rate for the high-risk form of the disease is 40–50%, and patients suffering metastatic recurrences have no known curative therapeutic options. Drugs targeted to specific genetic alterations in neuroblastoma may be more effective. Although neuroblastomas generally have few actionable genetic alterations at diagnosis, targetable mutations that confer therapy resistance may be selected for over time. Here, we analyzed cancer gene panel sequencing data from 151 neuroblastomas acquired at various time points during therapy to further define how the genomic landscape of neuroblastoma evolves. We found that relapsed tumors tended to have a higher frequency of mutations potentially targetable with currently available therapies, particularly in the RAS/MAPK pathway. Our data support the concept that therapeutic decisions targeting specific oncogenic mutations should be based on sequencing data obtained as close to the intervention as possible, and not be reliant on archived diagnostic material. Prospective clinical trials will be required to determine if sequencing data obtained at the time of tumor progression can lend to improved neuroblastoma patient outcomes.
PMCID: PMC5172533  PMID: 27997549
4.  Metastatic basal cell carcinoma with amplification of PD-L1: exceptional response to anti-PD1 therapy 
NPJ genomic medicine  2016;1:16037.
Metastatic basal cell carcinomas are rare malignancies harbouring Hedgehog pathway alterations targetable by SMO antagonists (vismodegib/sonidegib). We describe, for the first time, the molecular genetics and response of a patient with Hedgehog inhibitor-resistant metastatic basal cell carcinoma who achieved rapid tumour regression (ongoing near complete remission at 4 months) with nivolumab (anti-PD1 antibody). He had multiple hallmarks of anti-PD1 responsiveness including high mutational burden (> 50 mutations per megabase; 19 functional alterations in tissue next-generation sequencing (NGS; 315 genes)) as well as PDL1/PDL2/JAK2 amplification (as determined by both tissue NGS and by analysis of plasma-derived cell-free DNA). The latter was performed using technology originally developed for the genome-wide detection of sub-chromosomal copy-number alterations (CNAs) in noninvasive prenatal testing and showed numerous CNAs including amplification of the 9p24.3-9p22.2 region containing PD-L1, PD-L2 and JAK2. Of interest, PD-L1, PD-L2 and JAK2 amplification is a characteristic of Hodgkin lymphoma, which is exquisitely sensitive to nivolumab. In conclusion, selected SMO antagonist-resistant metastatic basal cell carcinomas may respond to nivolumab based on underlying molecular genetic mechanisms that include PD-L1 amplification and high tumour mutational burden.
PMCID: PMC5142752  PMID: 27942391
5.  Defects in DNA Repair Genes Predict Response to Neoadjuvant Cisplatin-based Chemotherapy in Muscle-invasive Bladder Cancer 
European urology  2015;68(6):959-967.
Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25–50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking.
To discover and validate biomarkers predictive of response to NAC for MIBC.
Design, setting, and participants
Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments–certified laboratory.
Outcome measurements and statistical analysis
The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set.
Results and limitations
Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p = 0.024) and validation (p = 0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p < 0.001; 87% sensitivity, 100% specificity) and better overall survival (p = 0.007). This test remained predictive for pathologic response in the validation set (p = 0.033), with a trend towards better overall survival (p = 0.055). These results require further validation in additional sample sets. Conclusions: Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin.
Patient summary
Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.
PMCID: PMC4764095  PMID: 26238431
Bladder cancer; Urothelial carcinoma; Biomarkers; Cisplatin sensitivity; Cisplatin resistance; ATM; RB1; FANCC; DNA repair; Neoadjuvant chemotherapy
6.  The distribution of BRAF gene fusions in solid tumors and response to targeted therapy 
International Journal of Cancer  2015;138(4):881-890.
Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in‐frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5′ fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall‐cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers.
What's new?
New results may help target a rare genetic alteration that promotes cancer. Activation of the BRAF gene is already known to spur tumor growth, and usually that activation results from a single amino acid substitution. BRAF‐inhibiting treatments, then, target that mutation. However, in some cases, BRAF gets revved up by a gene fusion. In our study, the authors tested 20,000 tumors and identified 55 BRAF gene fusions in 12 different tumor types. They found the gene fusions occurred more frequently in certain histologic subtypes, information which will help guide treatment strategies for patients with these tumor subtypes.
PMCID: PMC5049644  PMID: 26314551
cancer; solid tumors; BRAF fusions; pilocytic astrocytoma; pancreatic acinar carcinoma; Sptizoid melanoma; comprehensive genomic profiling; NGS; targeted therapy
7.  Comprehensive Genomic Profiling of Advanced Esophageal Squamous Cell Carcinomas and Esophageal Adenocarcinomas Reveals Similarities and Differences 
The Oncologist  2015;20(10):1132-1139.
Esophageal squamous cell carcinoma and esophageal adenocarcinoma are aggressive cancers with poor patient response to conventional chemotherapy and radiation treatment. In this study, comprehensive genomic profiling showed that the frequently altered genes and biological pathways differed between the two subtypes, and a high frequency of clinically relevant genomic alterations was noted as a means of finding a potential targeted therapy to be used in addition or as an alternative to conventional treatment.
Esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EACs) account for >95% of esophageal malignancies and represent a major global health burden. ESCC is the dominant histology globally but represents a minority of U.S. cases, with EAC accounting for the majority of U.S. cases. The patient outcomes for advanced ESCC and EAC are poor, and new therapeutic options are needed. Using a sensitive sequencing assay, we compared the genomic profiles of ESCC and EAC with attention to identification of therapeutically relevant genomic alterations.
Next-generation sequencing-based comprehensive genomic profiling was performed on hybridization-captured, adaptor ligation-based libraries to a median coverage depth of >650× for all coding exons of 315 cancer-related genes plus selected introns from 28 genes frequently rearranged in cancer. Results from a single sample were evaluated for all classes of genomic alterations (GAs) including point mutations, short insertions and deletions, gene amplifications, homozygous deletions, and fusions/rearrangements. Clinically relevant genomic alterations (CRGAs) were defined as alterations linked to approved drugs and those under evaluation in mechanism-driven clinical trials.
There were no significant differences by sex for either tumor type, and the median age for all patients was 63 years. All ESCCs and EACs were at an advanced stage at the time of sequencing. All 71 ESCCs and 231 EACs featured GAs on profiling, with 522 GAs in ESCC (7.4 per sample) and 1,303 GAs in EAC (5.6 per sample). The frequency of clinically relevant GAs in ESCC was 94% (2.6 per sample) and 93% in EAC (2.7 per sample). CRGAs occurring more frequently in EAC included KRAS (23% EAC vs. 6% ESCC) and ERBB2 (23% EAC vs. 3% ESCC). ESCC samples were enriched for CRGA in PIK3CA (24% ESCC vs. 10% EAC), PTEN (11% ESCC vs. 4% EAC), and NOTCH1 (17% ESCC vs. 3% EAC). Other GAs that differed significantly between histologic tumor types included SMAD4 (14% EAC vs. 1% ESCC), RB1 (14% ESCC vs. 2% EAC), SOX2 (18% ESCC vs. 1% EAC), and NFE2L2 (24% ESCC vs. 1% EAC).
ESCC and EAC share similarly high frequencies of overall and clinically relevant genomic alterations; however, the profiles of genomic alterations in the two diseases differ widely, with KRAS and ERBB2 far more frequently altered in EAC compared with ESCC and with mammalian target of rapamycin (MTOR) pathway genes (PIK3CA and PTEN) and NOTCH1 more frequently altered in ESCC compared with EAC. Comprehensive genomic profiling highlights the promise of identifying clinically relevant genomic alterations in both ESCC and EAC and suggests new avenues for molecularly directed therapies in esophageal cancer.
Implications for Practice:
Both esophageal squamous cell carcinoma and esophageal adenocarcinoma are aggressive cancers with poor patient response to conventional chemotherapy and radiation treatment. In this study, comprehensive genomic profiling was performed for 302 advanced esophageal cancers, and it was found that the frequently altered genes and biological pathways differed between the two subtypes. Also, a high frequency of clinically relevant genomic alterations was noted for both types of esophageal cancer as a means of finding a potential targeted therapy to be used in addition to or as an alternative to conventional treatment.
PMCID: PMC4591943  PMID: 26336083
Comprehensive genomic profiling; Next-generation sequencing; Esophageal cancer; Targeted therapy; Adenocarcinoma; Squamous cell
8.  Detection of crizotinib-sensitive lung adenocarcinomas with MET, ALK and ROS1 genomic alterations via comprehensive genomic profiling 
Clinical lung cancer  2015;16(5):e105-e109.
Crizotinib is an oral multitargeted tyrosine kinase inhibitor (TKI) with activity against lung cancers driven by ALK-rearrangements, ROS1-rearrangements and MET-amplification. Comprehensive genomic profiling (CGP) based on clinical next generation sequencing (NGS) can detect crizotinib-sensitive genomic changes. We describe use of CGP to identify tumors responsive to crizotinib.
Retrospective review of representative lung adenocarcinomas treated with crizotinib and assayed with a clinical NGS assay.
We report 3 cases of lung adenocarcinoma; one each identified to harbor an ALK-rearrangement (EML4-ALK), ROS1-rearrangement (SDC4-ROS1) and MET-amplification by genomic profiling. Notably, the MET-amplification was only detected by CGP as subsequent FISH testing did not show amplification. CGP also revealed other common genomic changes (somatic mutations [TP53 in 2 cases], deletions [CDKN2A in 1 case], amplifications [MCL1 in 1 case] and variants of unknown significance) in these cases. All patients received crizotinib 250 mg twice daily and achieved radiographic tumor reduction for months. The case harboring MET amplification of 10 copies achieved partial response and is one of the first MET-amplified lung cancer responsive to crizotinib in which the sole detection method was CGP.
CGP holds the promise of detecting predictive genomic alterations (somatic mutations, copy number changes and rearrangements) that may underlie tumor dependency in an oncogene and govern response to clinically-available TKIs for lung adenocarcinomas.
PMCID: PMC4418215  PMID: 25922291
mutation; lung cancer; next generation sequencing; genomic profiling; MET; ALK; ROS1; crizotinib
9.  Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged Non-Small Cell Lung Cancer with brain metastases 
In-frame fusion KIF5B (the-kinesin-family-5B-gene)-RET transcripts have been characterized in 1–2% of non-small cell lung cancers and are known oncogenic drivers. The RET tyrosine kinase inhibitor, vandetanib, suppresses fusion-induced, anchorage-independent growth activity. In vitro studies have shown that vandetanib is a high-affinity substrate of breast cancer resistance protein (Bcrp1/Abcg2) but is not transported by P-glycoprotein (P-gp), limiting its blood-brain barrier penetration. A co-administration strategy to enhance the brain accumulation of vandetanib by modulating P-gp/Abcb1- and Bcrp1/Abcg2-mediated efflux with mTOR inhibitors, specifically everolimus, was shown to increase the blood-brain barrier penetration. We report the first bench to bed-side evidence that RET inhibitor combined with an mTOR inhibitor is active against brain-metastatic RET-rearranged lung cancer and the first evidence of blood-brain barrier penetration. A 74 year old female with progressive adenocarcinoma of the lung (wild-type EGFR and no ALK rearrangement) presented for therapy options. A deletion of 5’RET was revealed by FISH assay, indicating RET-gene rearrangement. Because of progressive disease in the brain, she was enrolled in a clinical trial with vandetanib and everolimus (NCT01582191). Comprehensive genomic profiling revealed fusion of KIF5B (the-kinesin-family-5B-gene) and RET, in addition to AKT2 gene amplification. After 2 cycles of therapy a repeat MRI brain showed a decrease in the intracranial disease burden and PET /CT showed systemic response as well. Interestingly, AKT2 amplification seen is a critical component of the PI3K/mTOR pathway, alterations of which has been associated with both de novo and acquired resistance to targeted therapy. The addition of everolimus may have both overcome the AKT2 amplification to produce a response in addition to its direct effects on the RET gene. Our case report forms the first evidence of blood-brain-barrier penetration by vandetanib in combination with everolimus.Further research is required in this setting.
PMCID: PMC4998046  PMID: 25982012
RET; mTOR; Lung cancer; Brain metastases; Blood brain barrier; RET fusion; AKT; Next generation sequencing; Vandetanib; Everolimus; exceptional responder; outlier responder
10.  Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in “driver-negative” lung adenocarcinomas 
Broad, hybrid capture-based next-generation sequencing (NGS), as a clinical test, uses less tissue to identify more clinically relevant genomic alterations compared to profiling with multiple non-NGS tests. We set out to determine the frequency of such genomic alterations via this approach in tumors where previous extensive non-NGS testing had not yielded a targetable driver alteration.
We enrolled lung adenocarcinoma patients with a ≤15 pack-year smoking history whose tumors previously tested “negative” for alterations in 11 genes (mutations in EGFR, ERBB2, KRAS, NRAS, BRAF, MAP2K1, PIK3CA, and AKT1, and fusions involving ALK, ROS1, and RET) via multiple non-NGS methods. We performed hybridization capture of the coding exons of 287 cancer-related genes and 47 introns of 19 frequently rearranged genes and sequenced these to deep, uniform coverage.
Actionable genomic alterations with a targeted agent based on NCCN guidelines were identified in 26% (8/31: EGFR G719A, BRAF V600E, SOCS5-ALK, CLIP4-ALK, CD74-ROS1, KIF5B-RET [n=2], CCDC6-RET). 7 of these patients either received or are candidates for targeted therapy. Comprehensive genomic profiling using this method also identified a genomic alteration with a targeted agent available on a clinical trial in an additional 39% (12/31).
Broad, hybrid capture-based NGS identified actionable genomic alterations in 65% (95% CI 48–82%) of tumors from never or light smokers with lung cancers deemed without targetable genomic alterations by earlier extensive non-NGS testing. These findings support first-line profiling of lung adenocarcinomas using this approach as a more comprehensive and efficient strategy compared to non-NGS testing.
PMCID: PMC4917003  PMID: 25567908
next-generation sequencing; lung; adenocarcinoma; molecular profiling
11.  Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms 
Cancer discovery  2015;6(2):154-165.
Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of BRAFV600E mutations in ~50% of these patients provided the first molecular therapeutisc target in histiocytosis. However, recurrent driving mutations in the majority of BRAFV600E-wildtype, non-LCH patients are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in BRAFV600E-wildtype, non-LCH patients. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of MAP2K1- and ARAF-mutated, non-LCH patients using MEK and RAF inhibitors, respectively, resulted in clinical efficacy demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders.
PMCID: PMC4744547  PMID: 26566875
12.  Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors 
Oncoscience  2016;3(5-6):164-172.
There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway.
PMCID: PMC4965259  PMID: 27489863
breast cancer; FGFR; next-generation sequencing; PI3K
14.  Protein-Pacing and Multi-Component Exercise Training Improves Physical Performance Outcomes in Exercise-Trained Women: The PRISE 3 Study † 
Nutrients  2016;8(6):332.
The beneficial cardiometabolic and body composition effects of combined protein-pacing (P; 5–6 meals/day at 2.0 g/kg BW/day) and multi-mode exercise (resistance, interval, stretching, endurance; RISE) training (PRISE) in obese adults has previously been established. The current study examines PRISE on physical performance (endurance, strength and power) outcomes in healthy, physically active women. Thirty exercise-trained women (>4 days exercise/week) were randomized to either PRISE (n = 15) or a control (CON, 5–6 meals/day at 1.0 g/kg BW/day; n = 15) for 12 weeks. Muscular strength (1-RM bench press, 1-RM BP) endurance (sit-ups, SUs; push-ups, PUs), power (bench throws, BTs), blood pressure (BP), augmentation index, (AIx), and abdominal fat mass were assessed at Weeks 0 (pre) and 13 (post). At baseline, no differences existed between groups. Following the 12-week intervention, PRISE had greater gains (p < 0.05) in SUs, PUs (6 ± 7 vs. 10 ± 7, 40%; 8 ± 13 vs. 14 ± 12, 43% ∆reps, respectively), BTs (11 ± 35 vs. 44 ± 34, 75% ∆watts), AIx (1 ± 9 vs. −5 ± 11, 120%), and DBP (−5 ± 9 vs. −11 ± 11, 55% ∆mmHg). These findings suggest that combined protein-pacing (P; 5–6 meals/day at 2.0 g/kg BW/day) diet and multi-component exercise (RISE) training (PRISE) enhances muscular endurance, strength, power, and cardiovascular health in exercise-trained, active women.
PMCID: PMC4924173  PMID: 27258301
protein-pacing; exercise-trained women; PRISE; muscular fitness; augmentation index
15.  RICTOR amplification defines a novel subset of lung cancer patients who may benefit from treatment with mTOR1/2 inhibitors 
Cancer discovery  2015;5(12):1262-1270.
We identified amplification of RICTOR, a key component of the mTORC2, as the sole actionable genomic alteration in an 18-year-old never smoker with lung adenocarcinoma. It occurs in 13% of lung cancers (1016 cases) in TCGA and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTOR1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared to other agents targeting the PI3K/AKT/mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTOR1/2 inhibitors was observed. The index patient has been treated with mTOR1/2 inhibitors that led to tumor stabilization for over 18 months.
PMCID: PMC4670806  PMID: 26370156
RICTOR amplification; lung cancer; mTOR1/2 inhibitor
16.  Prospective Comprehensive Genomic Profiling of Advanced Gastric Carcinoma Cases Reveals Frequent Clinically Relevant Genomic Alterations and New Routes for Targeted Therapies 
The Oncologist  2015;20(5):499-507.
Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic gastric cancer (GC) cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies or targeted therapy-based clinical trials. Comprehensive genomic profiling of GC identified clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.
Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms.
Materials and Methods.
Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials.
Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor.
Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.
PMCID: PMC4425384  PMID: 25882375
Gastric cancer; Sequencing; Targeted therapy; Mutation; Profiling; MET
17.  EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib 
Cancer discovery  2015;5(11):1155-1163.
Oncogenic EGFR mutations are found in 10-35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). In analyzing the tumor from a 33-year-old male never smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18-25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD-transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI, afatinib. The patient eventually progressed, at which time, re-sequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target.
PMCID: PMC4631701  PMID: 26286086
Epidermal growth factor receptor (EGFR); non-small cell lung cancer; glioblastoma; sarcoma; next-generation sequencing; targeted therapy; intragenic; gene rearrangement; kinase domain; tyrosine kinase inhibitor (TKI); erlotinib; afatinib; AZD9291
18.  Lung Master Protocol (Lung-MAP)—A Biomarker-Driven Protocol for Accelerating Development of Therapies for Squamous Cell Lung Cancer: SWOG S1400 
The Lung Master Protocol (Lung-MAP, S1400) is a groundbreaking clinical trial designed to advance the efficient development of targeted therapies for squamous cell cancer (SCCA) of the lung. There are no approved targeted therapies specific to advanced lung SCCA, although The Cancer Genome Atlas (TCGA) project and similar studies have detected a significant number of somatic gene mutations/amplifications in lung SCCA, some of which are targetable by investigational agents. However, the frequency of these changes is low (5–20%), making recruitment and study conduct challenging in the traditional clinical trial setting. Here we describe our approach to development of a biomarker-driven phase 2/3 multi-substudy “Master Protocol,” employing a common platform (Next Generation DNA Sequencing) to identify actionable molecular abnormalities, followed by randomization to the relevant targeted therapy versus standard of care.
PMCID: PMC4654466  PMID: 25680375
19.  Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer 
The Oncologist  2015;20(3):316-322.
The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement in lung cancer is fluorescence in situ hybridization (FISH); however, FISH may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by immunohistochemistry should be strongly considered, and next-generation sequencing is recommended for borderline cases.
The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis.
Materials and Methods.
We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance.
Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC.
The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.
PMCID: PMC4350802  PMID: 25721120
EML4-ALK; Non-small cell lung cancer; Fluorescence in situ hybridization; Immunohistochemistry; Next-generation sequencing
20.  Loss of Heterozygosity at the CYP2D6 Locus in Breast Cancer: Implications for Germline Pharmacogenetic Studies 
Controversy exists regarding the impact of CYP2D6 genotype on tamoxifen responsiveness. We examined loss of heterozygosity (LOH) at the CYP2D6 locus and determined its impact on genotyping error when tumor tissue is used as a DNA source.
Genomic tumor data from the adjuvant and metastatic settings (The Cancer Genome Atlas [TCGA] and Foundation Medicine [FM]) were analyzed to characterize the impact of CYP2D6 copy number alterations (CNAs) and LOH on Hardy Weinberg equilibrium (HWE). Additionally, we analyzed CYP2D6 *4 genotype from formalin-fixed paraffin-embedded (FFPE) tumor blocks containing nonmalignant tissue and buccal (germline) samples from patients on the North Central Cancer Treatment Group (NCCTG) 89-30-52 tamoxifen trial. All statistical tests were two-sided.
In TCGA samples (n =627), the CYP2D6 LOH rate was similar in estrogen receptor (ER)–positive (41.2%) and ER-negative (35.2%) but lower in HER2-positive tumors (15.1%) (P < .001). In FM ER+ samples (n = 290), similar LOH rates were observed (40.8%). In 190 NCCTG samples, the agreement between CYP2D6 genotypes derived from FFPE tumors and FFPE tumors containing nonmalignant tissue was moderate (weighted Kappa = 0.74; 95% CI = 0.63 to 0.84). Comparing CYP2D6 genotypes derived from buccal cells to FFPE tumor DNA, CYP2D6*4 genotype was discordant in six of 31(19.4%). In contrast, there was no disagreement between CYP2D6 genotypes derived from buccal cells with FFPE tumors containing nonmalignant tissue.
LOH at the CYP2D6 locus is common in breast cancer, resulting in potential misclassification of germline CYP2D6 genotypes. Tumor DNA should not be used to determine germline CYP2D6 genotype without sensitive techniques to detect low frequency alleles and quality control procedures appropriate for somatic DNA.
PMCID: PMC4565524  PMID: 25490892
21.  Co-occurring genomic alterations define major subsets of KRAS - mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities 
Cancer discovery  2015;5(8):860-877.
The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma (LUAC) are poorly characterized. We performed an integrative analysis of genomic, transcriptomic and proteomic data from early-stage and chemo-refractory LUAC and identified three robust subsets of KRAS-mutant LUAC dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP) and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further reveal biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant LUAC with distinct biology and therapeutic vulnerabilities.
PMCID: PMC4527963  PMID: 26069186
KRAS; co-mutations; lung adenocarcinoma; STK11; HSP90
22.  Oncogenic Alterations in ERBB2/HER2 Represent Potential Therapeutic Targets Across Tumors From Diverse Anatomic Sites of Origin 
The Oncologist  2014;20(1):7-12.
The authors explored activating ERBB2 alterations in solid tumor specimens that underwent comprehensive genomic profiling using next-generation sequencing. Results showed that activating events in ERBB2/HER2 occurred across a wide variety of tumors. Additionally, standard slide-based tests for overexpression or amplification of ERBB2 would fail to detect the majority of activating mutations that occur overwhelmingly in the absence of copy number changes. Comprehensive genomic profiling of a more diverse set of tumor types may identify more patients likely to benefit from ERBB2-targeted therapy.
Targeted ERBB2/HER2 inhibitors are approved by the U.S. Food and Drug Administration for the treatment of breast, gastric, and esophageal cancers that overexpress or amplify HER2/ERBB2, as measured by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Activating mutations in ERBB2 have also been reported and are predicted to confer sensitivity to these targeted agents. Testing for these mutations is not performed routinely, and FISH and IHC are not applied outside of these approved indications.
Materials and Methods.
We explored the spectrum of activating ERBB2 alterations across a collection of ∼7,300 solid tumor specimens that underwent comprehensive genomic profiling using next-generation sequencing. Results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes.
Known oncogenic ERBB2 alterations were identified in tumors derived from 27 tissues, and ERBB2 amplification in breast, gastric, and gastroesophageal cancers accounted for only 30% of these alterations. Activating mutations in ERBB2 were identified in 131 samples (32.5%); amplification was observed in 246 samples (61%). Two samples (0.5%) harbored an ERBB2 rearrangement. Ten samples (2.5%) harbored multiple ERBB2 mutations, yet mutations and amplifications were mutually exclusive in 91% of mutated cases.
Standard slide-based tests for overexpression or amplification of ERBB2 would fail to detect the majority of activating mutations that occur overwhelmingly in the absence of copy number changes. Compared with current clinical standards, comprehensive genomic profiling of a more diverse set of tumor types may identify ∼3.5 times the number of patients who may benefit from ERBB2-targeted therapy.
PMCID: PMC4294606  PMID: 25480824
High-throughput nucleotide sequencing; Mutation; Antibodies; Monoclonal; Humanized; Trastuzumab; Lapatinib; Pertuzumab
24.  A metastatic colon adenocarcinoma harboring BRAF V600E has a durable major response to dabrafenib/trametinib and chemotherapy 
OncoTargets and therapy  2015;8:3561-3564.
The subset of metastatic colorectal adenocarcinomas that harbor BRAF V600E mutations are aggressive tumors with significantly shortened survival and limited treatment options. Here we present a colorectal cancer patient whose disease progressed through standard chemotherapy and who developed liver metastasis. Comprehensive genomic profiling (FoundationOne®) identified a BRAF V600E mutation in the liver lesion, as well as other genomic alterations consistent with colorectal cancers. Combination therapy of dabrafenib and trametinib with standard cytotoxic chemotherapy resulted in a durable major ongoing response for the patient. This report illustrates the utility of comprehensive genomic profiling with personalized targeted therapy for aggressive metastatic colorectal adenocarcinomas.
PMCID: PMC4671814  PMID: 26664139
oxaliplatin; colorectal adenocarcinoma; combination targeted therapy; BRAF mutations
25.  Identification of ANLN as ETV6 partner gene in recurrent t(7;12)(p15;p13): a possible role of deregulated ANLN expression in leukemogenesis 
Molecular Cancer  2015;14:197.
The ETV6 gene encodes an ETS family transcription factor that is involved in a myriad of chromosomal rearrangements found in hematological malignancies and other neoplasms. A recurrent ETV6 translocation, previously described in patients with acute myeloid leukemia (AML) (Genes Chromosomes Cancer 51:328–337,2012, Leuk Res 35:e212-214, 2011), whose partner has not been identified is t(7;12)(p15;p13). We herein report that the t(7;12)(p15;p13) fuses ETV6 to ANLN, a gene not previously implicated in the pathogenesis of hematological malignancies, and we demonstrate that this translocation leads to high expression of the fusion transcript in the myeloid and lymphoid lineages.
PMCID: PMC4653877  PMID: 26584717
Leukemia; Myeloid; Acute; Gene fusion; Translocation; Genetic

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