Publications

Featuring Archer technologies



Novel exon-exon breakpoint in CIC-DUX4 fusion sarcoma identified by anchored multiplex PCR (Archer FusionPlex Sarcoma Panel).

Journal of Clinical Pathology | January 2017

Benjamin Nathanae, Loke, Victor Kwan Min Lee, Jain Sudhanshi, Meng Kang Wong, Chik Hong Kuick, Mark Puhaindran, Kenneth Tou En Chang



Aims: We describe the clinical and pathological features and novel genetic findings of a case of CIC-DUX4 sarcoma occurring in the thigh of a 35-year-old man.

Methods: Fusion gene detection using a next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) was used to identify the novel fusion breakpoints of this CIC-DUX4 sarcoma using formalin-fixed and paraffin-embedded tumour material.

Results: This CIC-DUX4 sarcoma has a novel fusion breakpoint between exon 20 of the CIC gene and exon 1 of the DUX4 gene..

Conclusions: This case report describes an additional case of CIC-DUX4 sarcoma with a novel fusion breakpoint, and demonstrates the value of this next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) in both diagnosis for patient care and in identification of a novel fusion breakpoint in this tumour type.


The application of next generation-sequencing-based molecular diagnostics in endometrial stromal sarcoma.
Histopathology

Histopathology | October 2016 | Vol. 69(4): 551–559

Xiaodong Li, Mona Anand, Josh D Haimes, Namitha Manoj, Aaron M Berlin, Brian A Kudlow, Marisa R Nucci, Tony L Ng, Colin J R Stewart & Cheng-Han Lee


Aims: Endometrial stromal sarcomas (ESSs) are divided into low-grade and high-grade subtypes, with the latter showing more aggressive clinical behaviour. Although histology and immunophenotype can aid in the diagnosis of these tumours, genetic studies can provide additional diagnostic insights, as low-grade ESSs frequently harbour fusions involving JAZF1/SUZ12 and/or JAZF1/PHF1, whereas high-grade ESSs are defined by YWHAE–NUTM2A/B fusions. The aim of this study was to evaluate the utility of a next-generation sequencing (NGS)-based assay in identifying ESS fusions in archival formalin-fixed paraffin-embedded tumour samples.

Methods and results: We applied an NGS-based fusion transcript detection assay (Archer FusionPlex Sarcoma Panel) that targets YWHAE and JAZF1 fusions in a series of low-grade ESSs (n = 11) and high-grade ESSs (n = 5) that were previously confirmed to harbour genetic rearrangements by fluorescence in-situ hybridization (FISH) and/or reverse transcription polymerase chain reaction (RT-PCR) analyses. The fusion assay identified junctional fusion transcript sequences that corresponded to the known FISH/RT-PCR results in all cases. Four low-grade ESSs harboured JAZF1–PHF1 fusions with different junctional sequences, and all were correctly identified because of the open-ended nature of the assay design, using anchored multiplex polymerase chain reaction. Seven non-ESS sarcomas were also included as negative controls, and no strong ESS fusion candidates were identified in these cases.

Conclusions: Our findings demonstrate good sensitivity and specificity of an NGS-based gene fusion assay in the detection of ESS fusion transcripts.


Validation of a new algorithm for a quick and easy RT-PCR-based ALK test in a large series of lung adenocarcinomas: Comparison with FISH, immunohistochemistry and next generation sequencing assays.

Lung Cancer | September 2016 | Vol. 99: 11–16

Antonio Marchetti, Maria Vittoria Pace, Alessia Di Lorito, Sara Canarecci, Lara Felicioni, Tommaso D’Antuono, Marcella Liberatore, Giampaolo Filice, Luigi Guetti, Felice Mucilli, Fiamma Buttitta



Objectives: Anaplastic Lymphoma Kinase (ALK) gene rearrangements have been described in 3–5% of lung adenocarcinomas (ADC) and their identification is essential to select patients for treatment with ALK tyrosine kinase inhibitors. For several years, fluorescent in situ hybridization (FISH) has been considered as the only validated diagnostic assay. Currently, alternative methods are commercially available as diagnostic tests.

Material and Methods: A series of 217 ADC comprising 196 consecutive resected tumors and 21 ALK FISH-positive cases from an independent series of 702 ADC were investigated. All specimens were screened by IHC (ALK-D5F3-CDx-Ventana), FISH (Vysis ALK Break-Apart-Abbott) and RT-PCR (ALK RGQ RT-PCR-Qiagen). Results were compared and discordant cases subjected to Next Generation Sequencing.

Results: Thirty-nine of 217 samples were positive by the ALK RGQ RT-PCR assay, using a threshold cycle (Ct) cut-off ?35.9, as recommended. Of these positive samples, 14 were negative by IHC and 12 by FISH. ALK RGQ RT-PCR/FISH discordant cases were analyzed by the NGS assay with results concordant with FISH data. In order to obtain the maximum level of agreement between FISH and ALK RGQ RT-PCR data, we introduced a new scoring algorithm based on the ?Ct value. A ?Ct cut-off level ?3.5 was used in a pilot series. Then the algorithm was tested on a completely independent validation series. By using the new scoring algorithm and FISH as reference standard, the sensitivity and the specificity of the ALK RGQ RT-PCR(?Ct) assay were 100% and 100%, respectively.

Conclusions: Our results suggest that the ALK RGQ RT-PCR test could be useful in clinical practice as a complementary assay in multi-test diagnostic algorithms or even, if our data will be confirmed in independent studies, as a standalone or screening test for the selection of patients to be treated with ALK inhibitors.


The NAB2–STAT6 gene fusion in solitary fibrous tumor can be reliably detected by anchored multiplexed PCR for targeted next generation sequencing

Cancer Genetics | July 2016 | Vol. 209(7-8): 303–312

Natalya V. Guseva, Munir R. Tanas, Aaron A. Stence, Ramakrishna Sompallae, Jenna C. Schade, Aaron D. Bossler, Andrew M. Bellizzi, Deqin Ma'



Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic origin, which can affect any region of the body. 10–15% of SFTs metastasize and metastatic tumors are uniformly lethal with no effective therapies. The behavior of SFT is difficult to predict based on morphology. Recently, an intrachromosomal gene fusion between NAB2 and STAT6 was identified as the defining driving genetic event of SFT and different fusion types correlated with tumor histology and behavior. Due to the proximity of NAB2 and STAT6 on chromosome 12, this fusion may be missed by fluorescence in-situ hybridization. We evaluated 12 SFTs from 10 patients. All tumors showed strong nuclear staining for STAT6 by immunohistochemistry (IHC). The same formalin-fixed, paraffin-embedded blocks for IHC were used for gene fusion detection by a next-generation sequencing (NGS)-based assay. Targeted RNA fusion sequencing for gene fusions was performed using the Universal RNA Fusion Detection Kit, the Archer™ FusionPlex™ Sarcoma Panel and the Ion Torrent PGM, and data were analyzed using the Archer Analysis Pipeline 3.3. All tumors were positive for NAB2–STAT6 fusion. Six types of fusions were detected: NAB2ex4–STAT6ex2, NAB2ex2–STAT6ex5, NAB2ex6–STAT6ex16, NAB2ex6–STAT6ex17, NAB2ex3–STAT6ex18 and NAB2intron6–STAT6Ex17. The NGS findings were confirmed by RT-PCR followed by Sanger sequencing. No STAT6 fusion was detected in selected morphologic mimics of SFT. The assay also allows for detection of novel fusions and can detect NAB2–STAT6 fusions at a single-base resolution.


ALK Protein Analysis by IHC Staining after Recent Regulatory Changes: A Comparison of Two Widely Used Approaches, Revision of the Literature, and a New Testing Algorithm.
Journal of Thoracic Oncology

Journal of Thoracic Oncology | April 2016 | Vol. 11(4): 487–495

Antonio Marchetti, MD, PhD, Alessia Di Lorito, MD, Maria Vittoria Pace, BSc, Manuela Iezzi, MD, PhD, Lara Felicioni, PhD, Tommaso D’Antuono, B.Tech., Giampaolo Filice, PhD, Luigi Guetti, MD, Felice Mucilli, MD, Fiamma Buttitta, MD, PhD



Introduction: Recent regulatory changes have allowed the diagnostic use of immunohistochemical (IHC) analysis for the identification of patients with non–small cell lung cancer who are eligible for treatment with anaplastic lymphoma receptor tyrosine kinase (ALK) inhibitors. The U.S. Food and Drug Administration has approved the VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) as companion diagnostics, and the Italian Medicines Agency has recognized IHC analysis as a diagnostic test indicating an algorithm for patient selection.

Methods: On the basis of the new regulations, we compared two commonly used IHC assays on 1031 lung adenocarcinomas: the VENTANA ALK (D5F3) CDx Assay with the OptiView Amplification Kit (Ventana Medical Systems) and a standard IHC test with the clone 5A4 (Novocastra, Leica Biosystems, Newcastle Upon Tyne, United Kingdom) along with their interpretative algorithms. Fluorescence in situ hybridization (FISH) was performed in all cases. Next-generation sequencing was performed in FISH/IHC analysis–discordant samples.

Results: FISH gave positive results in 33 (3.2%) cases. When FISH was used as a reference, the VENTANA ALK (D5F3) CDx assay had a sensitivity of 90.9% ± 2.6%, a specificity of 99.8% ± 0.6%, and positive and negative predictive values of 93.8% ± 2.1% and 99.7% ± 0.6%, respectively. The clone 5A4–based IHC test showed a sensitivity of 90.9% ± 2.6%, a specificity of 98.3% ± 1.3%, and positive and negative predictive values of 63.8% ± 4.2% and 99.7% ± 0.6%, respectively. Five cases with IHC analysis/FISH-discordant results in our series were analyzed together with those previously reported in the literature. Overall, data from 35 patients indicate a response rate to ALK inhibitors in 100% of FISH-negative/IHC analysis–positive cases (seven of seven) and 46% of FISH-positive/IHC analysis–negative cases (13 of 28), respectively.

Conclusions: Our results confirm the difficulty in managing an IHC test without amplification in the absence of confirmatory FISH analysis, as well as the possibility of performing a direct diagnosis in approximately 90% of patients by the VENTANA ALK (D5F3) CDx Assay. On the basis of the recent regulatory changes, the data that have emerged from the literature, and the results of the present study, a new algorithm for ALK assessment in non–small cell lung cancer has been devised.


Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.
Applied Immunohistochemistry & Molecular Morphology

Applied Immunohistochemistry & Molecular Morphology | March 2016

Murphy, Danielle A. PhD; Ely, Heather A. MS; Shoemaker, Robert PhD; Boomer, Aaron MS; Culver, Brady P. PhD; Hoskins, Ian BS; Haimes, Josh D. BS; Walters, Ryan D. PhD; Fernandez, Diane MD; Stahl, Joshua A. PhD; Lee, Jeeyun MD; Kim, Kyoung-Mee MD; Lamoureux, Jennifer PhD; Christiansen, Jason PhD6



Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.


Next-Generation Sequencing and Fluorescence in Situ Hybridization Have Comparable Performance Characteristics in the Analysis of Pancrea

Gastrointestinal Endoscopy Journal | January 2016 | Vol. 83(1): 140–148

Martin Jones, MBBS, Zongli Zheng, MD, PhD, Jessica Wang, MD, Jonathan Dudley, MD, PhD, Emily Albanese, Abdurrahman Kadayifci, MD, Dora Dias-Santagata, PhD, Long Le, MD, William R. Brugge, MD, Carlos Fernandez-del Castillo, MD, Mari Mino-Kenudson, MD, A. John Iafrate, MD, PhD, Martha B. Pitman, MD,correspondence



Background and Aims: The value of next-generation sequencing (NGS) of pancreatic cyst fluid relative to the clinical and imaging impression has not been well-studied. The aim of this study was to assess the impact of NGS on the clinical diagnosis from imaging and carcinoembryonic antigen (CEA) and thus the management of pancreatic cysts.

Methods: Ninety-two pancreatic cyst fluids from 86 patients were analyzed by cytology, CEA, and targeted NGS. Cysts were classified by imaging as nonmucinous, mucinous, or not specified. NGS results were compared with the imaging impression stratified by CEA and cytology.

Results: NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels. The VHL gene in 2 intraductal papillary mucinous neoplasms (IPMNs) supported a serous cystadenoma. Twenty percent of cysts that were nonmucinous by imaging were mucinous by NGS. Of the 14 not-specific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%). A KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%. A KRAS mutation reclassified 19% of nonneoplastic cysts with nonmucinous CEA as mucinous. Seven cyst fluids (8%) had either a TP53 mutation or loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5 of 7 (71%) were clinically malignant, and high-grade cytology was detected in all 5. Overall, CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86% vs 57%).

Conclusions: NGS of pancreatic cyst fluid impacts clinical diagnosis and patient management by defining, supporting, or changing the clinical diagnosis based on imaging and CEA. NGS was most valuable in identifying mucinous cysts with nonmucinous CEA. An added benefit is the potential to detect mutations late in the progression to malignancy that may increase the risk classification of the cyst based on imaging and cytology.


Undifferentiated myxoid lipoblastoma with PLAG1-HAS2 fusion in an infant; morphologically mimicking primitive myxoid mesenchymal tumor of infancy (PMMTI)--diagnostic importance of cytogenetic and molecular testing and literature review.

Cancer Genetics | January 2016 | Vol. 209(1-2): 21–29

Mikako Warren, Brian K. Turpin, Melissa Mark, Teresa A. Smolarek, Xia Li'



Lipoblastoma is a benign myxoid neoplasm arising in young children that typically demonstrates adipose differentiation. It is often morphologically indistinguishable from primitive myxoid mesenchymal tumor of infancy (PMMTI), which is characterized by a well-circumscribed myxoid mass with a proliferation of primitive mesenchymal cells with mild cytologic atypia. PMMTI occurs in the first year of life and is known to have locally aggressive behavior. No specific genetic rearrangements have been reported to date. In contrast, the presence of PLAG1 (Pleomorphic Adenoma Gene 1) rearrangement is diagnostic for lipoblastoma. We hereby demonstrate the combined application of multiple approaches to tackle the diagnostic challenges of a rapidly growing neck tumor in a 3-month-old female. An incisional tumor biopsy had features of an undifferentiated, myxoid mesenchymal neoplasm mimicking PMMTI. However, tumor cells showed diffuse nuclear expression by immunohistochemical (IHC) stain. Conventional cytogenetic and fluorescence in situ hybridization (FISH) analyses as well as next generation sequencing (NGS) demonstrated evidence of PLAG1 rearrangement, confirming the diagnosis of lipoblastoma. This experience warrants that undifferentiated myxoid lipoblastoma can mimic PMMTI, and the combination of cytogenetic and molecular approaches is essential to distinguish these two myxoid neoplasms. Literature on lipoblastomas with relevant molecular and cytogenetic findings is summarized. Our case is the first lipoblastoma diagnosed with a PLAG1 fusion defined by NGS technology.


SEC31A-ALK Fusion Gene in Lung Adenocarcinoma

Cancer Research and Treatment | January 2016 | Vol. 48(1): 398–402

Kim RN, Choi YL, Lee MS, Lira ME, Mao M, Mann D, Stahl J, Licon A, Choi SJ, Van Vrancken M, Han J, Wlodarska I, Kim J



Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame. SEC31A-ALK encodes a predicted fusion protein of 1,438 amino acids comprising the WD40 domain of SEC31A at the N-terminus and ALK kinase domain at the C-terminus. Fluorescence in situ hybridization studies suggested that SEC31A-ALK was generated by an unbalanced genomic rearrangement associated with loss of the 3'-end of SEC31A. This is the first report of SEC31A-ALK fusion transcript in clinical NSCLC, which could be a novel diagnostic and therapeutic target for patients with NSCLC.


Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

British Journal of Cancer | December 2015 | Vol. 113: 1730–1734

Alessio Amatu, Alessio Somaschini, Giulio Cerea, Roberta Bosotti, Emanuele Valtorta, Pasquale Buonandi, Giovanna Marrapese, Silvio Veronese, David Luo, Zachary Hornby, Pratik Multani, Danielle Murphy, Robert Shoemaker, Calogero Lauricella, Laura Giannetta, Martina Maiolani, Angelo Vanzulli, Elena Ardini, Arturo Galvani, Antonella Isacchi, Andrea Sartore-Bianchi and Salvatore Siena



Background: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies.

Methods: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR.

Results: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response.

Conclusions: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.


Durable Clinical Response to Entrectinib in NTRK1-Rearranged Non-Small Cell Lung Cancer

Journal of Thoracic Oncology | December 2015 | Vol. 10: 1670–1674

Anna F. Farago, MD, PhD, Long P. Le, MD, PhD, Zongli Zheng, PhD, Alona Muzikansky, MA, Alexander Drilon, MD, Manish Patel, MD, Todd M. Bauer, MD, Stephen V. Liu, MD, Sai-Hong I. Ou, MD, PhD, David Jackman, MD, Daniel B. Costa, MD, PhD, Pratik S. Multani, MD, Gary G. Li, PhD, Zachary Hornby, MBA, Edna Chow-Maneval, PhD, David Luo, MPH, Jonathan E. Lim, MD, Anthony J. Iafrate, MD, PhD, Alice T. Shaw, MD, PhD




Introduction: Chromosomal rearrangements involving neurotrophic tyrosine kinase 1 (NTRK1) occur in a subset of non-small cell lung cancers (NSCLCs) and other solid tumor malignancies, leading to expression of an oncogenic TrkA fusion protein. Entrectinib (RXDX-101) is an orally available tyrosine kinase inhibitor, including TrkA. We sought to determine the frequency of NTRK1 rearrangements in NSCLC and to assess the clinical activity of entrectinib.

Methods: We screened 1378 cases of NSCLC using anchored multiplex polymerase chain reaction (AMP). A patient with an NTRK1 gene rearrangement was enrolled onto a Phase 1 dose escalation study of entrectinib in adult patients with locally advanced or metastatic tumors (NCT02097810). We assessed safety and response to treatment.

Results: We identified NTRK1 gene rearrangements at a frequency of 0.1% in this cohort. A patient with stage IV lung adenocrcinoma with an SQSTM1-NTRK1 fusion transcript expression was treated with entrectinib. Entrectinib was well tolerated, with no grade 3–4 adverse events. Within three weeks of starting on treatment, the patient reported resolution of prior dyspnea and pain. Restaging CT scans demonstrated a RECIST partial response (PR) and complete resolution of all brain metastases. This patient has continued on treatment for over 6 months with an ongoing PR.

Conclusions: Entrectinib demonstrated significant anti-tumor activity in a patient with NSCLC harboring an SQSTM1-NTRK1 gene rearrangement, indicating that entrectinib may be an effective therapy for tumors with NTRK gene rearrangements, including those with central nervous system metastases.


Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Journal of Molecular Diagnosis | November 2015 | Vol. 17(6): 661–668

Mia Y. Platt, Amir T. Fathi, Darrell R. Borger, Andrew M. Brunner, Robert P. Hasserjian, Leonora Balaj, Amy Lum, Stephen Yip, Dora Dias-Santagata, Zongli Zheng, Long P. Le, Timothy A. Graubert, A. John Iafrate, Valentina Nardic



Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications.


Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma

Clinical Chemistry | November 2015 | Vol. 61(11): 1408–1416

Jiaqi Huang, Johannes-Matthias Löhr, Magnus Nilsson, Ralf Segersvärd, Hans Matsson, Caroline Verbeke, Rainer Heuchel, Juha Kere, A. John Iafrate, Zongli Zheng and Weimin Ye1



Background: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease.

Methods: Patients with PDAC undergoing surgery from 2007 to 2012 (n 73) were followed from diag-nosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was val-idated by allele-specific PCR and digital PCR. We com-pared overall survival of patients according to KRAS mu-tation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types.

Results: The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500x). Low-frequency KRAS G12 mutations (allele frequency 1%–5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall sur-vival in T3-stage PDAC patients differed among KRAS mutation subtypes (P=0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2–27.8).

Conclusions: The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant sub-type G12V is associated with poorer survival, and that transversion variants are more common among smokers.


Unique Genetic and Survival Characteristics of Invasive Mucinous Adenocarcinoma of the Lung.

Journal of Thoracic Oncology | August 2015 | Vol 10(8): 1156–1162

Hyo Sup Shim, MD, PhD, Mari- Kenudson, MD, Zongli Zheng, PhD, Matthew Liebers, BSc, Yoon Jin Cha, MD, Quan Hoang Ho, BSc, Maristela Onozato, MD, PhD, Long Phi Le, MD, PhD, Rebecca S. Heist, MD, MPH, A. John Iafrate, PhD



Introduction: Invasive mucinous adenocarcinoma is a unique histologic subtype of lung cancer, and our knowledge of its genetic and clinical characteristics is rapidly evolving. Here, we present next- generation sequencing analysis of nucleotide variant and fusion events along with clinical follow-up in a series of lung mucinous adenocarcinoma.

Methods: We collected 72 mucinous adenocarcinomas from the United States and Korea. All had been previously assessed for KRAS and EGFR mutations. For KRAS wild-type cases (n = 30), we performed deep targeted next-generation sequencing for gene fusions and nucleotide variants and correlated survival and other clinical features.

Results: As expected, KRAS mutations were the most common alteration found (63% of cases); however, the distribution of nucleotide position alterations was more similar to that observed in gastrointestinal tumors than other lung tumors. Within the KRAS-negative cases, we found numerous potentially targetable gene fusions and mutations, including CD74-NRG1, VAMP2-NRG1, TRIM4-BRAF, TPM3-NTRK1, and EML4-ALK gene fusions and ERBB2, BRAF, and PIK3CA mutations. Unexpectedly, we found only two cases with TP53 mutation, which is much lower than observed in lung adenocarcinomas in general. The overall mutation burden was low in histologically confirmed mucinous adenocarcinomas from the public The Cancer Genome Atlas exome data set, regardless of smoking history, suggesting a link between TP53 status and mutation burden in mucinous tumors. There was no significant difference for recurrence-free survival between stage-matched mucinous and nonmucinous adenocarcinomas. It was notable that all recurrence sites were in the lungs for completely resected cases.

Conclusions: Our data suggest that mucinous adenocarcinoma is typified by (1) frequent KRAS mutations and a growing list of gene fusions, but rare TP53 mutations, (2) a low mutation burden overall, and (3) a recurrence-free survival similar to stage-matched nonmucinous tumors, with recurrences limited to the lungs.


Rearranging Detection of Gene Rearrangements.

Journal of Thoracic Oncology | August 2015 |Vol. 10(8): 1129-1130

Kurtis D. Davies, PhD, Robert C. Doebele, MD, PhD, Dara L. Aisner, MD, PhD



Recent years have witnessed a dramatic expansion in our knowledge of the genetic diversity of non–small-cell lung cancer (NSCLC), and in particular adenocarcinoma. This appreciation of the genetic landscape of the disease has resulted in the development of targeted therapies that are directed toward activated oncogenes. Several of these drugs have been approved for use in patients and many more are being investigated in clinical trials. However, there have been only been limited studies that have focused on histologic subtypes within NSCLC and more complete analyses of these specific disease entities are needed.


Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor.

Cancer Discovery | July 2015 | Vol. 5(7): 713–722

Zofia Piotrowska, Matthew J. Niederst, Chris A. Karlovich, Heather A. Wakelee, Joel W. Neal, Mari Mino-Kenudson, Linnea Fulton, Aaron N. Hata, Elizabeth L. Lockerman, Anuj Kalsy, Subba Digumarthy, Alona Muzikansky, Mitch Raponi, Angel R. Garcia, Hillary E. Mulvey, Melissa K. Parks, Richard H. DiCecca, Dora Dias-Santagata, A. John Iafrate, Alice T. Shaw, Andrew R. Allen, Jeffrey A. Engelman and Lecia V. Sequist



Rociletinib is a third-generation EGFR inhibitor active in lung cancers with T790M, the gatekeeper mutation underlying most first-generation EGFR drug resistance. We biopsied patients at rociletinib progression to explore resistance mechanisms. Among 12 patients with T790Mpositive cancers at rociletinib initiation, six had T790 wild-type rociletinib-resistant biopsies. Two T790 wild-type cancers underwent small cell lung cancer transformation; three T790M-positive cancers acquired EGFR amplification. We documented T790 wild-type and T790M-positive clones coexisting within a single pre-rociletinib biopsy. In fact, the pre-treatment fraction of T790M-positive cells impacted response to rociletinib. Longitudinal ctDNA analysis revealed an increase in plasma EGFR activating mutation and T790M heralded rociletinib resistance in some patients, while in others the activating mutation increased but T790M remained suppressed. Together, these findings demonstrate the role of tumor heterogeneity when therapies targeting a singular resistance mechanism are employed. To further improve outcomes, combination regimens that also target T790 wild-type clones are required.


Therapeutic targeting of anaplastic lymphoma kinase in lung cancer: a paradigm for precision cancer medicine.

Clinical Cancer Research | May 2015 | Vol. 21(10): 2227–2235

Ryohei Katayama, Christine M. Lovly and Alice T. Shaw



The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase was initially discovered as a component of the fusion protein nucleophosmin (NPM)–ALK in anaplastic large-cell lymphoma (ALCL). Genomic alterations in ALK, including rearrangements, point mutations, and genomic amplification, have now been identified in several malignancies, including lymphoma, non–small cell lung cancer (NSCLC), neuroblastoma, inflammatory myofibroblastic tumor, and others. Importantly, ALK serves as a validated therapeutic target in these diseases. Several ALK tyrosine kinase inhibitors (TKI), including crizotinib, ceritinib, and alectinib, have been developed, and some of them have already been approved for clinical use. These ALK inhibitors have all shown remarkable clinical outcomes in ALK-rearranged NSCLC. Unfortunately, as is the case for other kinase inhibitors in clinical use, sensitive tumors inevitably relapse due to acquired resistance. This review focuses on the discovery, function, and therapeutic targeting of ALK, with a particular focus on ALK-rearranged NSCLC. Clin Cancer Res; 21(10); 2227–35. ©2015 AACR.


Clinical Grade “SNaPshot” Genetic Mutation Profiling in Multiple Myeloma.

EBioMedicine | January 2015 | Vol. 2(1): 71–73

O'Donnell E, Mahindra A, Yee AJ, Nardi V, Birrer N, Horick N, Borger D, Finkelstein D, Iafrate JA, Raje N



Whole genome sequencing studies have identified several oncogenic mutations in multiple myeloma (MM). As MM progresses, it evolves genetically underscoring the need to have tools for rapid detection of targetable mutations to optimize individualized treatment. Massachusetts General Hospital (MGH) has developed a Clinical Laboratory Improvement Amendments (CLIA)-approved, high-throughput, genotyping platform to determine the mutation status of a panel of known oncogenes. Sequence analysis using SNaPshot on DNA extracted from bone marrow and extramedullary plasmacytomas is feasible and leads to the detection of potentially druggable mutations. Screening MM patients for somatic mutations in oncogenes may provide novel targets leading to additional therapies for this patient population


Anchored multiplex PCR for targeted next-generation sequencing.

Nature Medicine | December 2014 | Vol. 20: 1479–1484

Zongli Zheng, Matthew Liebers, Boryana Zhelyazkova, Yi Cao, Divya Panditi, Kerry D Lynch, Juxiang Chen, Hayley E Robinson, Hyo Sup Shim, Juliann Chmielecki, William Pao, Jeffrey A Engelman, A John Iafrate & Long Phi Le



We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5–100%) and 100% specificity (95% confidence limit: 99.3–100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.


Crizotinib in ROS1-rearranged non-small-cell lung cancer

New England Journal of Medicine | November 2014 | Vol. 371: 1963–1971

Alice T. Shaw, M.D., Ph.D., Sai-Hong I. Ou, M.D., Ph.D., Yung-Jue Bang, M.D., Ph.D., D. Ross Camidge, M.D., Ph.D., Benjamin J. Solomon, M.B., B.S., Ph.D., Ravi Salgia, M.D., Ph.D., Gregory J. Riely, M.D., Ph.D., Marileila Varella-Garcia, Ph.D., Geoffrey I. Shapiro, M.D., Ph.D., Daniel B. Costa, M.D., Ph.D., Robert C. Doebele, M.D., Ph.D., Long Phi Le, M.D., Ph.D., Zongli Zheng, Ph.D., Weiwei Tan, Ph.D., Patricia Stephenson, Sc.D., S. Martin Shreeve, M.D., Ph.D., Lesley M. Tye, Ph.D., James G. Christensen, Ph.D., Keith D. Wilner, Ph.D., Jeffrey W. Clark, M.D., and A. John Iafrate, M.D., Ph.D.



Background: Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non–small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.

Methods: We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase–polymerase-chain-reaction assays.

Results: The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.

Conclusions: In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.)


Identification of Oncogenic Mutations and Gene Fusions in the Follicular Variant of Papillary Thyroid Carcinoma.

The Journal of Clinical Endocrinology & Metabolism | November 2014 | Vol. 99(11): E2457–E2462

David G. McFadden, Dora Dias-Santagata, Peter M. Sadow, Kerry D. Lynch, Carrie Lubitz, Samuel E. Donovan, Zongli Zheng, Long Le, A. J. Iafrate, and Gilbert H. Daniels



Background: The diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) is in-creasingly common. Recent studies have suggested that FVPTC is heterogeneous and comprises multiple tumor types with distinct biological behaviors and underlying genetics.

Objective: The purpose of this work was to identify the prevalence of mutations and gene fusions in known oncogenes in a panel representative of the common spectrum of FVPTC diagnosed at an academic medical center and correlate the clinical and pathological features obtained at the initial diagnosis with the tumor genotype.

Materials and Methods: We performed SNaPshot genotyping on a panel of 129 FVPTCs of ?1cm for 90 point mutations or small deletions in known oncogenes and tumor suppressors and iden-tified gene fusions using an anchored multiplex PCR assay targeting a panel of rearranged oncogenes.

Results We identified a mutation or gene fusion in 70% (89 of 127) of cases. Mutations targeting the RAS family of oncogenes were the most frequently observed class of alterations, present in 36% (46 of 127) of cases, followed by BRAF mutation, present in 30% (38 of 127). We also detected oncogenic rearrangements not previously associated with FVPTC, including TFG-ALK and CREB3L2-PPAR?. BRAF mutation was significantly associated with unencapsulated tumor.

Conclusions: These data support the hypothesis that FVPTC is composed of distinct biological entities, with one class being identified by BRAF mutation and support the use of clinical geno-typing assays that detect a diverse array of rearrangements involving ALK and PPAR?. Additional studies are necessary to identify genetic drivers in the 30% of FVPTCs with no known oncogenic alteration and to better predict behavior in tumors with known genotypes.


A Novel Fusion of TPR and ALK in Lung Adenocarcinoma.

Journal of Thoracic Oncology | April 2014 | Vol. 9(4): 563–566

Yoon-La Choi, MD, PhD, Maruja E. Lira, BS, Mineui Hong, MD, Ryong Nam Kim, PhD, PhD Ryong Nam Kim, PhD Ryong Nam Kim, So-Jung Choi, PhD, Ji-Young Song, MS, Kinnari Pandy, MS, Derrick L. Mann, PhD, Joshua A. Stahl, PhD, Heather E. Peckham, PhD, Zongli Zheng, MD, PhD, Joungho Han, PhD, Mao Mao, MD, PhD, Jhingook Kim, MD, PhD



Introduction: Anaplastic lymphoma kinase (ALK) fusion is the most common mechanism for overexpression and activation in non–small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B, kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3, and huntingtin interacting protein 1 (HIP1).

Methods and Results: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression. By using an Anchored Multiplex polymerase chain reaction assay and sequencing, we found that tumor had a novel translocated promoter region (TPR)-ALK fusion. The fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (t(1;2)(q31.1;p23)). The TPR-ALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5’-2nd of the TPR and juxtamembrane and kinase domains encoded by the 3’-end of the ALK.

Conclusions: The novel fusion gene and its protein TRP-ALK, harboring coiled-coil and kinase domains, could possess transforming potential and responses to treatment with ALK inhibitors. This case is the first report of TPR-ALK fusion transcript in clinical tumor samples and could provide a novel diagnostic and therapeutic candidate target for patients with cancer, including non–small-cell lung carcinoma.


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