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Expanding the Spectrum of Genetic Alterations in Pseudomyogenic Hemangioendothelioma With Recurrent Novel ACTB-FOSB Gene Fusions
SurgPath-ACTB

The American Journal of Surgical Pathology | December, 2018

Agaram, Narasimhan P., MBBS; Zhang, Lei, MD; Cotzia, Paolo, MD; Antonescu, Cristina R., MD



Pseudomyogenic hemangioendothelioma (PHE) is an uncommon, rarely metastasizing vascular neoplasm with predilection to affect young adults. The tumors often present as multiple nodules involving various tissue planes, including superficial and deep soft tissues as well as bone. Recurrent SERPINE1-FOSB gene fusions have been reported as the hallmark genetic abnormality in PHE, however, in our experience, a number of cases with typical histology lack this genetic abnormality. In this study, we identify a novel ACTB-FOSB gene fusion, which is as prevalent as the initial translocation reported. We selected 15 consecutive cases of PHE with typical morphologic features which had material for molecular testing. The cohort included 10 males and 5 females, ranging in age from 17 to 58 years (median age: 33 y; mean age: 35.3 y). Eight (53%) cases were located in the lower extremities (foot, calf, tibia, thigh), 5 (33%) were located in the trunk, abdomen or pelvis (abdominal wall—2, shoulder, back, ischium) and 2 (13%) were located in the upper extremity (humerus and hand). Ten (67%) cases had multifocal presentation and 5 (33%) presented as solitary lesions. Three (20%) cases were located only in the superficial dermis and subcutaneous tissues, 4 (27%) involved the superficial and deep soft tissue and 8 (53%) cases involved only the deep soft tissue and bone. Using fluorescence in situ hybridization and ARCHER fusionplex analysis we identified a novel ACTB-FOSB gene fusion in 7 cases, while the remaining 8 had the previously described SERPINE1-FOSB fusion. The clinicopathologic features and behavior of PHE associated with the ACTB-FOSB gene fusion were similar to those harboring the SERPINE1-FOSB; except that tumors with the ACTB variant were more often associated with solitary presentation. In conclusion, our results expand the spectrum of genetic alterations in PHE with a novel gene fusion identified in half of the cases. We speculate that some of the novel targeted therapies that have shown promise in SERPINE1-FOSB-positive PHE might also be beneficial in this molecular subset.


High-grade myoepithelial carcinoma can show histologically undifferentiated/anaplastic features
ADP-Myo

Annals of Diagnostic Pathology | December, 2018

Yingting Mok, Abbas Agaimy, Shi Wang, Chik Hong Kuick, Kenneth Tou-En Chang, Fredrik Petersson



High grade malignant tumors with a poorly-/un- differentiated morphology pose significant diagnostic challenges. Increasingly, the use of adjunct immunohistochemical and molecular tests to characterize and delineate the histopathologic phenotype of these tumors has become necessary, particularly in head and neck tumors. Recently, several entities with a poorly-/un- differentiated light microscopic morphology have been defined based on specific immunohistochemical and genetic characteristics. We herein describe two cases of high-grade myoepithelial carcinoma, one occurring in the submandibular gland and the other occurring in the left nasal cavity, both showing undifferentiated histological and anaplastic cytomorphological features. This led to very broad differential diagnostic considerations and the diagnosis was only established after extensive immunohistochemical studies. Molecular testing for HPV was negative in both cases. Gene fusion analysis using a targeted sequencing assay (Archer® FusionPlex® system) did not identify fusions involving PLAG1, HMGA2, EWSR1 or ALK genes in either case. The submandibular tumor showed an aggressive clinical course, with diffuse pulmonary metastases at presentation, whilst the nasal cavity tumor showed only localized disease. Awareness of a subcategory of high-grade myoepithelial carcinomas with undifferentiated light microscopical features is of significant importance in antibody selection for immunohistochemical investigation of poorly- /undifferentiated malignant tumors in the head and neck region. This histological variant of myoepithelial carcinoma adds to the growing list of differential diagnoses in this diagnostically complex and multifaceted field.


Diagnosis of known sarcoma fusions and novel fusion partners by targeted RNA sequencing with identification of a recurrent ACTB-FOSB fusion in pseudomyogenic hemangioendothelioma
Modern Pathology

Modern Pathology | November, 2018

Guo Zhu, Ryma Benayed, Caleb Ho, Kerry Mullaney, Purvil Sukhadia, Kelly Rios, Ryan Berry, Brian P. Rubin, Khedoudja Nafa, Lu Wang, David S. Klimstra, Marc Ladanyi & Meera R. Hameed



Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of sarcomas. We have validated and implemented a clinical molecular diagnostic assay, MSK- Fusion Solid, for detection of gene fusions in solid tumors, including sarcomas. Starting with RNA extracted from formalin-fixed paraffin-embedded tumor material, this targeted RNA sequencing assay utilizes anchored multiplex PCR to detect oncogenic fusion transcripts involving 62 genes known to be recurrently rearranged in solid tumors including sarcomas without prior knowledge of fusion partners. From 1/2016 to 1/2018, 192 bone and soft tissue tumors were submitted for MSK- Fusion Solid analysis and 96% (184/192) successfully passed all the pre-sequencing quality control parameters and sequencing steps. These sarcomas encompass 24 major tumor types, including 175 soft tissue tumors and 9 osteosarcomas. Ewing and Ewing-like sarcomas, rhabdomyosarcoma, and sarcoma-not otherwise specified were the three most common tumor types. Diagnostic in-frame fusion transcripts were detected in 43% of cases, including 3% (6/184) with novel fusion partners, specifically TRPS1-PLAG1, VCP-TFE3, MYLK-BRAF, FUS-TFCP2, and ACTB-FOSB, the latter in two cases of pseudomyogenic hemangioendothelioma, representing a novel observation in this sarcoma. Our experience shows that this targeted RNA sequencing assay performs in a robust and sensitive fashion on RNA extracted from most routine clinical specimens of sarcomas thereby facilitating precise diagnosis and providing opportunities for novel fusion partner discovery.


Molecular Profiling of Salivary Gland Intraductal Carcinoma Revealed a Subset of Tumors Harboring NCOA4-RET and Novel TRIM27-RET Fusions – A Report of 17 Cases
SurgPath-NCOA4

The American Journal of Surgical Pathology | November, 2018

Alena Skálová, Tomas Vanecek, Emmanuelle Uro-Coste, Justin A. Bishop, Ilan Weinreb, Lester D.R. Thompson, Stefano de Sanctis, Marco Schiavo-Lena, Jan Laco, Cécile Badoual, Thalita Santana Conceiçao, Nikola Ptáková, Martina Baněčkova, Marketa Miesbauerová, Michal Michal



Intraductal carcinoma (IC) is the new World Health Organization designation for tumors previously called “low-grade cribriform cystadenocarcinoma” and “low-grade salivary duct carcinoma.” The relationship of IC to salivary duct carcinoma is controversial, but they now are considered to be distinct entities. IC is a rare low-grade malignant salivary gland neoplasm with features similar to mammary atypical ductal hyperplasia or ductal carcinoma in situ, that shows diffuse S100 protein and mammaglobin positivity and is only partially defined genetically. (Mammary analogue) secretory carcinoma harboring ETV6-NTRK3, and in rare cases ETV6-RET fusion, shares histomorphologic and immunophenotypical features with IC. Recently, RET rearrangements and NCOA4-RET have been described in IC, suggesting a partial genetic overlap with mammary analogue secretory carcinoma. Here, we genetically characterize the largest cohort of IC to date to further explore this relationship. Seventeen cases of IC were analyzed by next-generation sequencing using the FusionPlex Solid Tumor kit (ArcherDX). Identified fusions were confirmed using fluorescence in situ hybridization break apart and, in some cases, fusion probes, and a reverse transcription polymerase chain reaction designed specifically to the detected breakpoints. All analyzed cases were known to be negative for ETV6 rearrangement by fluorescence in situ hybridization and for ETV6-NTRK3 fusion by reverse transcription polymerase chain reaction. Next-generation sequencing analysis detected a NCOA4-RET fusion transcript joining exon 7 or 8 of NCOA4 gene and exon 12 of RET gene in 6 cases of intercalated duct type IC; and a novel TRIM27-RET fusion transcript between exons 3 and 12 in 2 cases of salivary gland tumors displaying histologic and immunohistochemical features typical of apocrine IC. A total of 47% of IC harbored a fusion involving RET. In conclusion, we have confirmed the presence of NCOA4-RET as the dominant fusion in intercalated duct type IC. A novel finding in our study has been a discovery of a subset of IC patients with apocrine variant IC harboring a novel TRIM27-RET.


Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor
MCT-ctDNA

Molecular Cancer Therapeutics | November, 2018

Heidi M. Namløs, Kjetil Boye, Skyler J. Mishkin, Tale Barøy, Susanne Lorenz, Bodil Bjerkehagen, Eva W. Stratford, Else Munthe, Brian A. Kudlow, Ola Myklebost and Leonardo A. Meza-Zepeda



Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing tumor-specific aberrations through noninvasive sampling. In gastrointestinal stromal tumor (GIST), analysis of KIT and PDGFRA mutations is important for therapeutic decisions, but the invasiveness of traditional biopsies limits the possibilities for repeated sampling. Using targeted next-generation sequencing, we have analyzed circulating cell-free DNA from 50 GIST patients. Tumor-specific mutations were detected in 16 of 44 plasma samples (36%) from treatment-naïve patients and in three of six (50%) patients treated with tyrosine kinase inhibitors. A significant association between detection of ctDNA and the modified National Institutes of Health risk classification was found. All patients with metastatic disease had detectable ctDNA, and tumor burden was the most important detection determinant. Median tumor size was 13.4 cm for patients with detectable mutation in plasma compared with 4.4 cm in patients without detectable mutation (P = 0.006). ctDNA analysis of a patient with disease progression on imatinib revealed that multiple resistance mutations were synchronously present, and detailed analysis of tumor tissue showed that these were spatially distributed in the primary tumor. Plasma samples taken throughout the course of treatment demonstrated that clonal evolution can be monitored over time. In conclusion, we have shown that detection of GIST-specific mutations in plasma is particularly feasible for patients with high tumor burden. In such cases, we have demonstrated that mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the whole tumor, and may guide treatment decisions during progression.


Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples
Journal of Thoracic Oncology

Journal of Thoracic Oncology | October, 2018

Kurtis D. Davies, PhD, Anh T. Le, BA, Jamie Sheren, PhD, Hala Nijmeh, PhD, Katherine Gowan, BS, Kenneth L. Jones, PhD, Marileila Varella-Garcia, PhD, Dara L. Aisner, MD, PhD, Robert C. Doebele, MD, PhD



Introduction

ROS1 gene fusions are a well-characterized class of oncogenic driver found in approximately 1% to 2% of NSCLC patients. ROS1-directed therapy in these patients is more efficacious and is associated with fewer side effects compared to chemotherapy and is thus now considered standard-of-care for patients with advanced disease. Consequently, accurate detection of ROS1 rearrangements/fusions in clinical tumor samples is vital. In this study, we compared the performance of three common molecular testing approaches on a cohort of ROS1 rearrangement/fusion-positive patient samples.

Methods

Twenty-three ROS1 rearrangement/fusion-positive clinical samples were assessed by at least two of the following molecular testing methodologies: break-apart fluorescence in situ hybridization, DNA-based hybrid capture library preparation followed by next-generation sequencing (NGS), and RNA-based anchored multiplex polymerase chain reaction library preparation followed by NGS.

Results

None of the testing methodologies demonstrated 100% sensitivity in detection of ROS1 rearrangements/fusions. Fluorescence in situ hybridization results were negative in 2 of 20 tested samples, the DNA-based NGS assay was negative in 4 of 18 tested samples, and the RNA-based NGS assay was negative in 3 of 19 tested samples. For all three testing approaches, we identified assay characteristics that likely contributed to false-negative results. Additionally, we report that genomic breakpoints are an unreliable predictor of breakpoints at the transcript level, likely due to alternative splicing.

Conclusions

ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.


A novel case of an aggressive superficial spindle cell sarcoma in an adult resembling fibrosarcomatous dermatofibrosarcoma protuberans and harboring an EML4‐NTRK3 fusion
Journal of Cutaneous Pathology

Journal of Cutaneous Pathology | September, 2018

Nicholas Olson, Omid Rouhi, Linsheng Zhang, Christina Angeles, Julia Bridge, Dolores Lopez‐Terrada, Thomas Royce, Konstantinos Linos



A subset of soft tissue sarcomas often harbors recurrent fusions involving protein kinases. While some of these fusion events have shown utility in arriving at a precise diagnosis, novel fusions in otherwise difficult to classify sarcomas continue to be identified. We present a case of a 40‐year‐old female who noted a lower back nodule in 2010 that was initially labeled as a dermatofibrosarcoma protuberans with fibrosarcomatous transformation. The lesion recurred the following year and metastasized to the groin 6 years later. Because of some morphologic peculiarities, molecular characterization was pursued in the metastatic focus, which revealed the neoplasm was negative for the COL1A1‐PDGFB fusion. However, anchored multiplex polymerase chain reaction for targeted next‐generation sequencing (Archer Dx) detected an EML4‐NTRK3 fusion, which was confirmed by reverse transcription‐PCR, Sanger sequencing and RNA sequencing analysis of the recurrent and metastatic specimens. Although various soft tissue neoplasms involving fusions with NTRK genes are well‐reported, the current case could not be easily classified in any of the established entities. Nevertheless, it raises interesting questions regarding the importance of classification, prognosis, and treatment for some of these tyrosine kinase fusion‐driven sarcomas.


Clinical Evaluation of Massively Parallel RNA Sequencing for Detecting Recurrent Gene Fusions in Hematologic Malignancies
JMD-Heme

The Journal of Molecular Diagnostics | Published online: September, 2018

Borahm Kim, Hyeonah Lee, Saeam Shin, Saeam ShinEmail the author Saeam Shin, Seung-Tae Lee, Seung-Tae LeeEmail the author Seung-Tae Lee, Jong Rak Choi



The application of next-generation sequencing (NGS) technology in clinical diagnostics should proceed with care. We have evaluated the clinical validity of two commercially available RNA fusion panels, the TruSight RNA fusion panel (Illumina) and FusionPlex Pan-Heme Kit (ArcherDx), to detect recurrent translocations in hematologic malignancies. Twenty-four bone marrow samples taken at the initial diagnosis of patients with acute leukemia and chronic myeloid leukemia were included. To assess the limit of detection, serial dilutions of BCR-ABL1 (e1a2) positive RNAs were prepared using a commercial reference material. Both NGS panels detected 19 cases with recurrent translocations identified with reverse transcription-PCR, as well as a case with KMT2A-AFF1 with false-negative results in reverse transcription-PCR. Two rare translocations, DDX3X-MLLT10 and NUP98-HOXC13, were additionally identified using NGS panels. The detection limit ranged from 10-1 to 10-2, which was not satisfactory for samples with low tumor burden. To conclude, RNA fusion panels were suitable for the initial diagnosis, however, for follow-up samples, conventional reverse transcription-PCR should be selected.


Extra-osseous Ewing sarcoma of the pancreas: case report with radiologic, pathologic, and molecular correlation, and brief review of the literature
Vir-Ewing

Virchows Archiv | September, 2018

Miglena K. Komforti, Evgeniya Sokolovskaya, Catherine A. D’Agostino, Ryma Benayed, Rebecca M. Thomas



In 2002, due to extensive histomorphologic, immunohistochemical, and cytogenetic similarities, the World Health Organization unified undifferentiated small round blue cell neoplasms of soft tissue and bone (previously segregated as Ewing sarcoma or Primitive Neuroectodermal tumor) into one category: Ewing family of tumors (EFT). Osseous EFT are more common, and while extra-osseous EFT can occur anywhere in the body, those of the pancreas are rare and likely to be seen in the second decade of life in the head of the pancreas. We report the case of a 39-year-old Caucasian male with a large heterogeneously enhancing mass in the pancreatic body. Pathologic examination showed a malignant round blue cell tumor diffusely positive for CD99, chromogranin, and synaptophysin; Ki-67 proliferation index was greater than 80%. FISH showed EWSR1 gene rearrangement in 90% of cells and Archer FusionPlexTM-targeted RNA sequencing analysis identified the EWSR1-FLI1 fusion transcript. The diagnosis of EFT of the pancreas was rendered. Unfortunately, the patient had minimal improvement and was transitioned to oral pain medications to continue care at a different institution.


Novel gene fusions in secretory carcinoma of the salivary glands: enlarging the ETV6 family
Hum-Path

Human Pathology | September, 2018

Julie Guilmette MD; Dora Dias-Santagata PhD; Vania Nosé MD PhD; Jochen K. Lennerz MD PhD; Peter M. Sadow MD PhD



Secretory carcinoma (SC) of the salivary gland is a low-grade malignancy associated with a well-defined clinical, histologic, immunohistochemical, and cytogenetic signature. Although the t(12;15) (p13;q25) translocation resulting in an ETV6-NTRK3 gene fusion is well-documented, advances in molecular profiling in salivary gland tumors have led to the discovery of RET as another ETV6 gene fusion partner in SC. Here, we applied an RNA-based next-generation sequencing (NGS) approach for fusion detection on 14 presumed SC. The cases included seven secretory carcinomas with classic ETV6- NTRK3 gene fusion and three secretory carcinomas harboring ETV6-RET gene fusion. In addition, two cases revealed a NCOA4-RET gene fusion and were subsequently reclassified as intraductal carcinomas. One case with an unusual dual pattern morphology revealed a novel translocation involving ETV6, NTRK3 and MAML3 gene rearrangements. Interestingly, no ETV6-NTRK3 or ETV6-RET SC were ever documented to have this unique dual pattern morphology or harbor a MAML3 mutation. The remaining case had no detected chromosomal abnormalities. Advances in molecular profiling of SC has led to the discovery of novel fusion partners such as RET and now MAML3. Further molecular characterization of salivary gland neoplasms is needed as these mutations may present alternative therapeutic targets in patients with these tumors.


Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing
JMD-st

The Journal of Molecular Diagnostics | September, 2018

Suk Wai Lam, Anne-Marie Cleton-Jansen, Arjen H.G. Cleven, Dina Ruano, Tom van Wezel, Karoly Szuhai,y and Judith V.M.G. Bovée



Molecular assays for translocation detection in bone and soft tissue tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. In this study, the applicability of a novel technique termed anchored multiplex PCR (AMP) for next-generation sequencing (NGS), using the Archer FusionPlex Sarcoma kit, aimed at 26 genes, was evaluated and compared with FISH and reverse transcriptase-PCR. In case of discrepant results, further analysis occurred with a third independent technique. Eighty-one samples were subjected to AMP-based targeted NGS, and 86% (n = 70) were successfully conducted and were either fusion positive (n = 48) or fusion negative, but met all criteria for good quality (n = 22). A concordance of 90% was found between NGS and conventional techniques. AMP-based targeted NGS showed superior results, as in four cases reverse transcriptase-PCR and FISH were false negative. Moreover, because the assay targets one partner of a gene fusion, novel or rare fusion partners can be identified. Indeed, it revealed COL1A1 and SEC31A as novel fusion partners for USP6 in nodular fasciitis. Despite the fact that fusions involving genes outside the selectively captured region cannot be detected and false-negative results due to poor quality samples can be encountered, this method has demonstrated excellent diagnostic utility for translocation detection in sarcomas.


NCOA4-RET fusion in colorectal cancer: Therapeutic challenge using patient-derived tumor cell lines
JC-NCORET

Journal of Cancer | July, 2018

Sun Young Kim, Seiyoon Oh Oh, Kyung Kim, Jeeyun Lee, SoYoung Kang, Kyoung-Mee Kim, WooYong Lee, Seung Tae Kim, Dohyun Nam Nam



The RET fusion is considered as the potential novel target in solid tumors. However, RET fusion is not well yet identified in colorectal cancer (CRC), and the effect of RET kinase inhibitor is also not evaluated in CRC with RET fusion. We established patient-derived tumor cells (PDCs) with RET fusion from recurrent brain metastatic lesion that newly appeared during the surveillance for stage III CRC patient. To investigate therapeutic options to CRC patient with a RET fusion, we performed cell viability assays using the PDCs. NCOA4-RET fusion was detected by FusionPlex using the resected brain metastatic tissue of CRC patient with solitary brain metastasis and then reconfirmed by fluorescence in situ hybridization (FISH) test. We also confirmed the RET fusions by a qPCR in matched PDCs. We tested whether the PDCs from RET fusion colon cancer were sensitive to carbozantinib, sorafenib, vandetanib, and PD0331992. Cell viability assays showed that carbozantinib, sorafenib, and PD0332991 did not suppress cell viability. Only, vandetanib revealed the significant inhibitory effect in MTT proliferation assay. Next, we analyzed regulation of targeted downstream pathways upon exposure to vandetanib by immunoblot assay. In colon cancer PDCs with NCOA4-RET fusion, vandetanib potently inhibited AKT and ERK phosphorylation. This study shows that vandetanib might be one of useful treatment strategies for CRC patient with NCOA4-RET fusion. Therefore, inhibition of the RET kinase is a promising targeted therapy for cancer patients whose tumors harbor a RET rearrangement.


Clinicopathologic Features of Non–Small-Cell Lung Cancer Harboring an NTRK Gene Fusion
JCO Precision Oncology

Journal of Clinical Oncology Precision Oncology | July, 2018

Anna F. Farago, Martin S. Taylor, Robert C. Doebele, Viola W. Zhu, Shivaani Kummar, Alexander I. Spira, Theresa A. Boyle, Eric B. Haura, Maria E. Arcila, Ryma Benayed, Dara L. Aisner, Nora K. Horick, Jochen K. Lennerz, Long P. Le, A. John Iafrate, Sai-Hong I. Ou, Alice T. Shaw, Mari Mino-Kenudson, and Alexander Drilon



Purpose

Gene rearrangements that involve NTRK1/2/3 can generate fusion oncoproteins that contain the kinase domains of TRKA/B/C, respectively. These fusions are rare in non–small-cell lung cancer (NSCLC), with frequency previously estimated to be < 1%. Inhibition of TRK signaling has led to dramatic responses across tumor types with NTRK fusions. Despite the potential benefit of identifying these fusions, the clinicopathologic features of NTRK fusion-positive NSCLCs are not well characterized.

Methods

We compiled a database of patients with NSCLCs that harbor NTRK fusions. We characterized clinical, molecular, and histologic features with central review of histology.

Results

We identified 11 patients with NSCLCs that harbor NTRK gene fusions verified by next-generation sequencing and with available clinical and pathologic data. Fusions involved NTRK1 (n = 7) and NTRK3 (n = 4), with five and two distinct fusion partners, respectively. Fifty-five percent of cohort patients were male with a median age at diagnosis of 47.6 years (range, 25.3 to 86.0 years) and a median smoking history of 0 pack-years (range, 0 to 58 pack-years). Seventy-three percent of patients had metastatic disease at diagnosis. No concurrent alterations in KRAS, EGFR, ALK, ROS1, or other known oncogenic drivers were identified. Nine patients had adenocarcinoma, including two with invasive mucinous adenocarcinoma and one with adenocarcinoma with neuroendocrine features; one had squamous cell carcinoma; and one had neuroendocrine carcinoma. By collating data on 4,872 consecutively screened, unique patients with NSCLC, we estimate a frequency of NTRK fusions in NSCLC of 0.23% (95% CI, 0.11% to 0.40%).

Conclusion

NTRK fusions occur in NSCLCs across sexes, ages, smoking histories, and histologies. Given the potent clinical activity of TRK inhibitors, we advocate that all NSCLCs be screened for NTRK fusions by using a multiplexed next-generation sequencing–based fusion assay.


Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples
PLOS-extract

PLOS ONE | May 17, 2018

Stine H. Kresse, Heidi M. Namløs, Susanne Lorenz, Jeanne-Marie Berner, Ola Myklebost, Bodil Bjerkehagen, Leonardo A. Meza-Zepeda



Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.


The ETV6-RET Gene Fusion is Found in ETV6-rearranged Low-grade Sinonasal Adenocarcinoma Without NTRK3 Involvement
SurgPathETV-RET

The American Journal of Surgical Pathology | Published online: April 20, 2018

Simon Andreasen, Katalin Kiss, Linea C. Melchior, Jan Laco

Targetable Gene Fusions Associate With the IDH Wild-Type Astrocytic Lineage in Adult Gliomas
Neuropath-Glioma

Journal of Neuropathology and Experimental Neurology | April, 2018

Sherise D Ferguson, MD Shouhao Zhou, PhD Jason T Huse, MD, PhD John F de Groot, MD Joanne Xiu, PhD Deepa S Subramaniam, MD Shwetal Mehta, PhD Zoran Gatalica, MD Jeffrey Swensen, PhD Nader Sanai, MD David Spetzler, MS, PhD, MBA Amy B Heimberger, MD



Gene fusions involving oncogenes have been reported in gliomas and may serve as novel therapeutic targets. Using RNA-sequencing, we interrogated a large cohort of gliomas to assess for the incidence of targetable genetic fusions. Gliomas (n = 390) were profiled using the ArcherDx FusionPlex Assay. Fifty-two gene targets were analyzed and fusions with preserved kinase domains were investigated. Overall, 36 gliomas (9%) harbored a total of 37 potentially targetable fusions, the majority of which were found in astrocytomas (n = 34). Within this lineage 11% (25/235) of glioblastomas, 12% (5/42) of anaplastic astrocytomas, 8% (2/25) of grade II astrocytomas, and 33% (2/6) of pilocytic astrocytoma harbored targetable fusions. Fusions were significantly more frequent in IDH wild-type tumors (12%, n = 31/261) relative to IDH mutants (4%; n = 4/109) (p = 0.011). No fusions were seen in oligodendrogliomas. The most frequently observed therapeutically targetable fusions were in FGFR (n = 12), MET (n = 11), and NTRK (n = 8). Several additional novel fusions that have not been previously described in gliomas were identified including EGFR:VWC2 and FGFR3:NBR1. In summary, targetable gene fusions are enriched in IDH wild-type high-grade astrocytic tumors, which will influence enrollment in and interpretation of clinical trials of glioma patients.


Molecular Minimal Residual Disease Monitoring in Acute Myeloid Leukemia: Challenges and Future Directions
JMD-MRD

The Journal of Molecular Diagnostics | Published online: April 21, 2018

Adrian Selim and Andrew S. Moore



The ability to sensitively monitor minimal residual disease (MRD) has played a key role in improving the management and outcomes for a number of leukemias, particularly acute promyelocytic leukemia and childhood acute lymphoblastic leukemia. In contrast, MRD monitoring in acute myeloid leukemia (AML) has been limited by variable assay methodologies and a relative paucity of patient-specific MRD markers. Inter and intratumor genetic heterogeneity pose significant challenges for the identification of molecular markers suitable for MRD monitoring in AML, particularly for those cases without structural chromosomal rearrangements associated with fusion genes. Furthermore, the need to discriminate which mutations may be suitable for MRD monitoring creates additional complexity. The mainstay of current molecular MRD monitoring is real-time quantitative PCR, targeting fusion genes, mutations, and gene overexpression. New technologies, particularly next-generation sequencing approaches, offer new ways to overcome these limitations. Here, we review the techniques available for molecular MRD monitoring in AML and discuss their utility in clinical practice.


Molecular Profiling of Mammary Analog Secretory Carcinoma Revealed a Subset of Tumors Harboring a Novel ETV6-RET Translocation: Report of 10 Cases
SurgPathol

The American Journal of Surgical Pathology | February, 2018

Skalova A, Vanecek T, Martinek P, Weinreb I, Stevens TM, Simpson RHW, Hyrcza M, Rupp NJ, Baneckova M, Michal M Jr, Slouka D, Svoboda T, Metelkova A, Etebarian A, Pavelka J, Potts SJ, Christiansen J, Steiner P, Michal M



ETV6 gene abnormalities are well described in tumor pathology. Many fusion partners of ETV6 have been reported in a variety of epithelial, mesenchymal, and hematological malig- nancies. In salivary gland tumor pathology, however, the ETV6-NTRK3 translocation is specific for (mammary analog) secretory carcinoma, and has not been documented in any other salivary tumor type. The present study comprised a clinical, histologic, and molecular analysis of 10 cases of secretory carcinoma, with typical morphology and immunoprofile har- boring a novel ETV6-RET translocation.


Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia
MCR

Molecular Cancer Research | February, 2018

Sadia Afrin, Christine R.C. Zhang, Claus Meyer, Caedyn L. Stinson, Thy Pham, Timothy J.C. Bruxner, Nicola C. Venn, Toby N. Trahair, Rosemary Sutton, Rolf Marschalek, J. Lynn Fink and Andrew S. Moore



Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the “gold standard” technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10−7 and transcripts down to 0.005 copies/ng.


Primary Renal Hybrid Low-grade Fibromyxoid Sarcoma-Sclerosing Epithelioid Fibrosarcoma: An Unusual Pediatric Case With EWSR1-CREB3L1 Fusion.
PedDevPath

Pediatric and Develepmental Pathology | February 9, 2018

Mok Y, Pang YH, Sanjeev JS, Kuick CH, Chang KT



Low-grade fibromyxoid sarcoma (LGFMS) and sclerosing epithelioid fibrosarcoma (SEF) are rare tumors with distinct sets of morphological features, both characterized by MUC4 immunoreactivity. Tumors exhibiting features of both entities are considered hybrid LGFMS-SEF lesions. While the majority of LGFMS cases are characterized by FUS-CREB3L2 gene fusions, most cases of pure SEF show EWSR1 gene rearrangements. In the largest study of hybrid LGFMS-SEF tumors to date, all cases exhibited FUS rearrangements, a similar genetic profile to LGFMS. We herein describe the clinicopathological features and genetic findings of a case of primary renal hybrid LGFMS-SEF occurring in a 10-year-old child, with disseminated metastases. Fusion gene detection using a next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) was performed on both the primary renal tumor that showed the morphology of a LGFMS, and a cervical metastasis that showed the morphology of SEF. An EWSR1-CREB3L1 gene fusion occurring between exon 11 of EWSR1 and exon 6 of CREB3L1 was present in both the LGFMS and SEF components. This unusual case provides evidence that a subset of hybrid LGFMS-SEF harbor EWSR1-CREB3L1 gene fusions. In this case, these features were associated with an aggressive clinical course, with disease-associated mortality occurring within 12 months of diagnosis.


Simultaneous Detection of Single-Nucleotide Variant, Deletion/Insertion, and Fusion in Lung and Thyroid Carcinoma Using Cytology Specimen and an RNA-Based Next-Generation Sequencing Assay.
Cancer Cytopathology

Cancer Cytopathology | January 24, 2018

Guseva NV, Jaber O, Stence AA, Sompallae K, Bashir A, Sompallae R, Bossler AD, Jensen CS, Ma D



BACKGROUND: Molecular testing for epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion is routinely performed in patients with stage IV lung adenocarcinoma to assess their eligibility for targeted therapy. Fine-needle aspiration (FNA)-derived material frequently is the only pathologic material available. The identification of genomic aberrations in thyroid nodules from FNA smears may help stratify cancer risk and spare patients from a second surgery. In the current study, the authors tested nucleic acid extracted from the cytology smears of lung and thyroid carcinomas for simultaneous detection of single-nucleotide variant, insertion/deletion, and gene fusion using an RNA-based next-generation sequencing assay. METHODS: A total of 27 cases (17 lung and 10 thyroid carcinomas, the majority of which had known variants) were tested. Areas of interest were scrapped from stained smears using a scalpel. Total nucleic acid was extracted. Gene fusion and mutational analysis was performed using the Comprehensive Thyroid and Lung FusionPlex Assay. Data were analyzed using the analysis pipeline provided by the vendor. Eleven cases with available formalin-fixed, paraffin-embedded (FFPE) tissue were tested in parallel. RESULTS: Gene fusions were detected in 6 cases; common single-nucleotide variants in EGFR, RAS, and BRAF in 14 cases; and in-frame deletions within EGFR in 3 cases. A concord- ance rate of 100% was observed between FNA and FFPE tissue. CONCLUSIONS: Cytology preparations can be a reliable source for the detection of both DNA and RNA aberrations. The ability to simultaneously detect multiple types of genomic variants is crucial for patients with advanced cancer and maximizes the usefulness of cytology specimens.


Comprehensive Molecular Profiling of Advanced/Metastatic Olfactory Neuroblastomas
PLoS-Neuroblast

PLOS ONE | January 11, 2018

Jasmina Topcagic, Rebecca Feldman, Anatole Ghazalpour, Jeffrey Swensen, Zoran Gatalica, Semir Vranic



Olfactory neuroblastoma (ONB) is a rare, locally aggressive, malignant neoplasm originating in the olfactory epithelium in the nasal vault. The recurrence rate of ONB remains high and there are no specific treatment guidelines for recurrent/metastatic ONBs. This study retrospectively evaluated 23 ONB samples profiled at Caris Life Sciences (Phoenix, Arizona) using DNA sequencing (Sanger/NGS [Illumina], n = 15) and gene fusions (Archer FusionPlex, n = 6), whole genome RNA microarray (HumanHT-12 v4 beadChip, Illumina, n = 4), gene copy number assays (chromogenic and fluorescent in situ hybridization), and immunohistochemistry. Mutations were detected in 63% ONBs including TP53, CTNNB1, EGFR, APC, cKIT, cMET, PDGFRA, CDH1, FH, and SMAD4 genes. Twenty-one genes were over-expressed and 19 genes under-expressed by microarray assay. Some of the upregulated genes included CD24, SCG2, and IGFBP-2. None of the cases harbored copy number variations of EGFR, HER2 and cMET genes, and no gene fusions were identified. Multiple protein biomarkers of potential response or resistance to classic chemotherapy drugs were identified, such as low ERCC1 [cisplatin sensitivity in 10/12], high TOPO1 [irinotecan sensitivity in 12/19], high TUBB3 [vincristine resistance in 13/14], and high MRP1 [multidrug resistance in 6/6 cases]. None of the cases (0/10) were positive for PD-L1 in tumor cells. Overexpression of pNTRK was observed in 67% (4/6) of the cases without underlying genetic alterations. Molecular alterations detected in our study (e.g., Wnt and cKIT/PDGFRA pathways) are potentially treatable using novel therapeutic approaches. Identified protein biomarkers of response or resistance to classic chemotherapy could be useful in optimizing existing chemotherapy treatment(s) in ONBs.


ALK Gene Fusions in Epithelioid Fibrous Histiocytoma: A Study of 14 Cases, With New Histopathological Findings.
Dermatopathol

The American Journal of Dermatopathology | Published online: January 11, 2018

Kazakov DV, Kyrpychova L, Martinek P, Grossmann P, Steiner P, Vanecek T, Pavlovsky M, Bencik, Michal M, Michal M



Previous studies showed that ALK is often positive in epithelioid fibrous histiocytoma (EFH). Two cases of EFH with ALK gene fusions have been recorded. Our objective was to study a series of EFH to present histopathological variations of EFH, identify novel ALK gene fusions, and determine whether there is a correlation between histopathological features and particular gene. We investigated 14 cases of EFH, all ALK immunopositive. The cases were assessed histopathologically as well as for ALK and TFE-3 rearrangements using FISH and ALK gene fusions using next-generation sequencing. The analysis of the sequencing results was performed using the Archer Analysis software (v5; ArcherDX Inc). The study group consisted of 8 female and 6 male patients, ranging in age from 18 to 79 years (mean 42 years; median 37.5 years). All presented with a solitary lesion. Microscopically, most lesions were polypoid and composed of epithelioid cells with ample cytoplasm. In addition, a variable number of bi-, tri-, or multinucleated, spindled, multilobated, cells with eccentric nuclei, cells with nuclear pseudoinclusions, mucinous, and grooved cells were admixed. In 5 cases, the predominant epithelioid cell component consisted of rather small cells, whereas spindled cells dominated in 3 cases. Of these, 2 lesions were composed rather of pale eosinophilic to clear cells, occasioning a resemblance to PEComa or leiomyoma. Immunohistochemically, all cases expressed ALK and 11 were positive for TFE-3. The break apart test for ALK was positive in 11 cases, whereas specimens from the remaining 3 cases were not analyzable. ALK genes fusions were found in all but 3 cases and included SQSTM1-ALK (3), VCL-ALK (3), TMP3-ALK (2), PRKAR2A-ALK (1), MLPH-ALK (1), and EML4-ALK (1). No correlation between histological features and type of ALK fusion was found. TFE-3 break apart test was negative. It is concluded that ALK-immunopositive EFH shows ALK gene fusions that involve various protein-coding genes, implicated in a variety of biological processes. Rare variants of EFH rather consist of spindled “non-epithelioid” cells.


Recurrent EML4–NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy.
Modern Pathology

Modern Pathology | November 3, 2017

Alanna J Church, Monica L Calicchio, Valentina Nardi, Alena Skalova, Andre Pinto, Deborah A Dillon, Carmen R Gomez-Fernandez, Namitha Manoj, Josh D Haimes, Joshua A Stahl, Filemon S Dela Cruz, Sarah Tannenbaum-Dvir, Julia L Glade-Bender, Andrew L Kung, Steven G DuBois, Harry P Kozakewich, Katherine A Janeway, Antonio R Perez-Atayde and Marian H Harris



Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6–NTRK3 gene fusion. However, a number of case reports have identified variant fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4–NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6–NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4–NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4–NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4–NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.


Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches.
Pathobiology

Scientific Reports | Published online: October 2, 2017

Julie A. Vendrell, Sylvie Taviaux, Benoît Béganton, Sylvain Godreuil, Patricia Audran, David Grand, Estelle Clermont, Isabelle Serre, Vanessa Szablewski, Peter Coopman, Julien Mazières, Valérie Costes, Jean-Louis Pujol, Pierre Brousset, Isabelle Rouquette & Jérôme Solassol



Rearrangements of the anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) represent a novel molecular target in a small subset of tumors. Although ALK rearrangements are usually assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), molecular approaches have recently emerged as relevant alternatives in routine laboratories. Here, we evaluated the use of two different amplicon-based next-generation sequencing (NGS) methods (AmpliSeq and Archer®FusionPlex®) to detect ALK rearrangements, and compared these with IHC and FISH. A total of 1128 NSCLC specimens were screened using conventional analyses, and a subset of 37 (15 ALK-positive, and 22 ALK-negative) samples were selected for NGS assays. Although AmpliSeq correctly detected 25/37 (67.6%) samples, 1/37 (2.7%) and 11/37 (29.7%) specimens were discordant and uncertain, respectively, requiring further validation. In contrast, Archer®FusionPlex® accurately classified all samples and allowed the correct identification of one rare DCTN1-ALK fusion, one novel CLIP1-ALK fusion, and one novel GCC2-ALK transcript. Of particular interest, two out of three patients harboring these singular rearrangements were treated with and sensitive to crizotinib. These data show that Archer®FusionPlex® may provide an effective and accurate alternative to FISH testing for the detection of known and novel ALK rearrangements in clinical diagnostic settings.


Primary Benign and Malignant Thyroid Neoplasms With Signet Ring Cells: Cytologic, Histologic, and Molecular Features.
American Journal of Clinical Pathology

American Journal of Clinical Pathology | September 1, 2017

Nada A Farhat, Ayse M Onenerk, Jeffrey F Krane, Dora Dias-Santagata, Peter M Sadow, William C Faquin



Objectives: Signet ring cells (SRCs) can be seen in a variety of thyroid tumors and can pose a diagnostic pitfall on cytology. This study describes the cytologic, histomorphologic, and molecular aspects of a cohort of primary thyroid tumors with SRCs.

Methods: A search was performed of the Massachusetts General Hospital and Brigham and Women’s Hospital (Boston, MA) pathology archives for the keywords thyroid, signet, and signet ring features between 2000 and 2014. Seven thyroidectomy specimens with corresponding thyroid fine-needle aspiration (FNA) were obtained. Cytology and histopathology slides were evaluated. Molecular analysis was performed using anchored multiplex polymerase chain reaction (AMP).

Results: The cohort consisted of four follicular adenomas (FAs), two noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs), and one secretory carcinoma (SC). The FNA diagnoses were atypia of undetermined significance (n = 3), suspicious for follicular neoplasm (n = 3), and suspicious for malignancy (n = 1). Molecular analyses revealed PTEN and FGFR3 mutations in an FA and NIFTP, respectively, and an ETV6-NTRK3 fusion in a case of primary thyroid gland SC.

Conclusions: Our study demonstrates the range of thyroid tumors with SRCs. While most thyroid tumors with SRCs are benign, primary thyroid SC should also be considered in the differential diagnosis.


Dramatic Response to Crizotinib in a Patient With Lung Cancer Positive for an HLA-DRB1-MET Gene Fusion.
Pathobiology

Pathobiology | Published online: August 29, 2017

Kurtis D. Davies, Terry L. Ng, Adriana Estrada-Bernal, Anh T. Le, Peter R. Ennever, D. Ross Camidge



Non–small-cell lung cancer (NSCLC) is a disease in which tumor growth is commonly driven by alterations along the receptor tyrosine kinase–RAS-RAF–mitogen-activated protein kinase pathway. Consequently, activated kinases along this axis represent attractive therapeutic targets. Activation of kinases can occur via multiple mechanisms at the genetic level, including mutation, amplification, and rearrangement/fusion with other genes. The two best-characterized gene fusion classes in NSCLC are those that involve ALK and ROS1—accounting for approximately 5% and 2% of patients, respectively—and inhibition of these fusion proteins is now a standard of care. Gene fusions that involve RET, NTRK1/2/3, BRAF, FGFR1/2/3, EGFR, and NRG1 have also been identified in lung cancer samples—an approximately 2% frequency for RET, < 1% for others—and strategies to target these aberrations are in development.

Crizotinib, the first US Food and Drug Administration–approved drug for patients with NSCLC who harbor ALK rearrangements and currently the only approved drug for patients with NSCLC who harbor ROS1 rearrangements was originally designed to be an inhibitor of the MET gene product hepatocyte growth factor receptor (HGFR) (c-Met). Activation of HGFR-mediated signaling—most commonly via MET amplification and/or MET mutations that result in exon 14 skipping—is well described in NSCLC, and crizotinib is currently being investigated in these settings. Recently, gene fusions that involve MET have been described in various cancers, including NSCLC, and a pediatric patient with glioblastoma who harbored a MET fusion was reported to have achieved a partial response to crizotinib. In this study, we report the first case of a MET fusion in lung cancer identified and treated during course of clinical care and the dramatic response of the patient’s tumor to crizotinib treatment.


Role of Next-Generation Sequencing as a Diagnostic Tool for the Evaluation of Bone and Soft-Tissue Tumors.
Pathobiology

Pathobiology | Published online: August 18, 2017

Szurian, Kinga; Kashofer, Karl; Liegl-Atzwanger, Bernadette



Bone and soft-tissue tumors are in general rare. Diagnosing these tumors is challenging based on the significant number of different tumor entities, the rareness of these tumors, and the considerable morphological heterogeneity which can be found within a single tumor entity. Considering that more than half of the described soft-tissue tumors and approximately 25% of the bone tumors harbor recurrent genetic alterations, the use of auxiliary molecular examinations should be strongly considered. Molecular analyses are important to confirm the diagnosis, to guide treatment, to provide information about prognosis, and to allow patient recruitment for basket trials based on the molecular signature of a tumor. In addition, novel molecular alterations detected by next-generation sequencing (NGS) obtain further insights into the pathogenesis of these rare tumors and allow a more detailed genetic classification. Based on our single-center results of NGS using the Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher Scientific) for mutational analyses as well as the Archer FusionPlex Sarcoma Kit (ArcherDX, Inc) to detect gene fusions in 26 genes since early 2016, we have experienced NGS as a very sensitive method to detect genetic alterations. In our experience, the use of the Archer FusionPlex Sarcoma Kit is superior to fluorescent in situ hybridization as an auxiliary tool in the routine workup of soft-tissue and bone tumor.


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 | Published online: March 2016 | Print: August 2017 | Vol. 25(7): 513-523

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.


USP6 activation in nodular fasciitis by promoter-swapping gene fusions.
Modern Pathology

Modern Pathology | July 28 2017 | Vol. 30: 1577-1588

Nimesh R Patel, John SA Chrisinger, Elizabeth G Demicco, Stephen F Sarabia, Jacquelyn Reuther, Erica Kumar, Andre M Oliveira, Steven D Billings, Judith VMG Bovee, Angshumoy Roy, Alexander J Lazar, Dolores H Lopez-Terrada and Wei-Lien Wang



Nodular fasciitis is a self-limited myofibroblastic lesion that can be misdiagnosed as a sarcoma as a result of its rapid growth, cellularity, and sometimes prominent mitotic activity. A recurrent translocation t(17;22) has been identified in nodular fasciitis, fusing the coding region of USP6 to the promoter region of MYH9, and resulting in increased USP6 expression. A subset of cases show USP6 rearrangement without the typical fusion variants by RT-PCR, or any MYH9 rearrangement by FISH. We sought to further characterize such tumors using molecular diagnostic assays. A novel RT-PCR assay was designed to detect the two known MYH9–USP6 fusion types in formalin-fixed paraffin-embedded and frozen tissue, and a break-apart FISH assay was designed to detect USP6 rearrangement. Twenty-six cases of nodular fasciitis diagnosed between 2002 and 2013 were retrieved from the pathology files of our institutions and were confirmed to be positive by FISH and/or RT-PCR. Seven samples showed USP6 rearrangement by FISH but were negative for MYH9–USP6 fusion by RT-PCR; these cases were subjected to a next-generation sequencing assay utilizing anchored multiplex PCR technology. This assay targets a single partner gene associated with fusions in bone and soft tissue tumors for agnostic detection of gene fusion partners. Novel fusion partners were identified in all seven cases and confirmed by RT-PCR. Structurally, all fusions consisted of the juxtaposition of the entire coding region of USP6 with the promoter of the partner gene, driving increased USP6 expression. This study confirms the neoplastic nature of nodular fasciitis, defines additional pathogenic fusion partners, and adds to the growing body of literature on USP6- associated neoplasia. Given the diagnostic challenges of these tumors, molecular assays can be useful ancillary tools; however, the prevalence of promoter swapping must be recognized when interpreting results.


Uterine Inflammatory Myofibroblastic Tumors Frequently Harbor ALK Fusions With IGFBP5 and THBS1.

The American Journal of Surgical Pathology | June 2017 | Vol. 41(6): 773-780

Haimes, Josh D. BSc; Stewart, Colin J.R. FRCPA; Kudlow, Brian A. PhD; Culver, Brady P. PhD; Meng, Bo PhD; Koay, Eleanor FRCPA; Whitehouse, Ann FRCPA; Cope, Nichola MBChB, MRCPath; Lee, Jen-Chieh MD, PhD; Ng, Tony MD, PhD; McCluggage, W. Glenn FRCPath; Lee, Cheng-Han PhD, MD



Inflammatory myofibroblastic tumor (IMT) can occur in a number of anatomic sites, including the uterus. Like its soft tissue counterpart, uterine IMT frequently expresses ALK and harbors ALK genetic rearrangements. The aim of this study is to fully characterize the genetic fusions that occur in uterine IMT. We studied 11 uterine IMTs with typical histology and 8 uterine myxoid smooth muscle tumors (5 leiomyomas, 1 smooth muscle tumor of uncertain malignant potential, and 2 leiomyosarcomas) in which the differential of IMT was considered, using a RNA-sequencing–based fusion assay to detect genetic fusions involving ALK, ROS1, RET, NTRK1/3, and other genes. ALK was expressed in 10 of 11 IMTs and 1 tumor initially categorized as a myxoid leiomyoma (granular cytoplasmic staining with paranuclear accentuation). Fusion transcripts involving ALK were identified in 9 of 10 ALK immunopositive IMTs, with 3 harboring IGFBP5-ALK, 3 harboring THBS1-ALK, 2 harboring FN1-ALK, and 1 harboring TIMP3-ALK. Among the smooth muscle tumors, IGFBP5-ALK fusion transcript was identified in only 1 ALK immunopositive case. Further review revealed that although a diagnosis of IMT was considered for the ALK immunopositive myxoid leiomyoma, this diagnosis was not initially rendered only because fluorescence in situ hybridization analysis was interpreted as negative for ALK genetic rearrangement; this case is best reclassified as an IMT. Notably, all the ALK fusions identified in our study included the transmembrane domain-encoding exon 19 of ALK. Our findings confirm the high frequency of ALK fusions in uterine IMT, with an enrichment of novel 5? ALK fusion partners (IGFBP5, THBS1, and TIMP3) and exon 19-containing ALK fusion. Given that IGFBP5 and FN1 are both situated on the same chromosome as ALK, fluorescence in situ hybridization analysis for ALK rearrangement may not be reliable and a negative result should not exclude a diagnosis of uterine IMT if the histologic features and ALK immunostaining findings are supportive.


Identification of NTRK3 Fusions in Childhood Melanocytic Neoplasms.

The Journal of Molecular Diagnostics | May 2017 | Vol. 19(3): 387-396

Lu Wang, Klaus J. Busam, Ryma Benayed, Robert Cimera, Jiajing Wang, Ryan Denley, Mamta Rao, Ruth Aryeequaye, Kerry Mullaney, Long Cao, Marc Ladanyi, and Meera Hameed



Spitzoid neoplasms are a distinct group of melanocytic tumors. Genetically, they lack mutations in common melanoma-associated oncogenes. Recent studies have shown that spitzoid tumors may contain a variety of kinase fusions, including ROS1, NTRK1, ALK, BRAF, and RET fusions. We report herein the discovery of recurrent NTRK3 gene rearrangements in childhood melanocytic neoplasms with spitzoid and/or atypical features, based on genome-wide copy number analysis by single-nucleotide polymorphism array, which showed intragenic copy number changes in NTRK3. Break-apart fluorescence in situ hybridization confirmed the presence of NTRK3 rearrangement, and a novel MYO5A-NTRK3 transcript, representing an in-frame fusion of MYO5A exon 32 to NTRK3 exon 12, was identified using a rapid amplification of cDNA ends-based anchored multiplex PCR assay followed by next-generation sequencing. The predicted MYO5A-NTRK3 fusion protein consists of several N-terminal coiled-coil protein dimerization motifs encoded by MYO5A and C-terminal tyrosine kinase domain encoded by NTRK3, which is consistent with the prototypical structure of TRK oncogenic fusions. Our study also demonstrates how array-based copy number analysis can be useful in discovering gene fusions associated with unbalanced genomic aberrations flanking the fusion points. Our findings add another potentially targetable kinase fusion to the list of oncogenic fusions in melanocytic tumors.


Cytological, molecular, and clinical features of noninvasive follicular thyroid neoplasm with papillary-like nuclear features versus invasive forms of follicular variant of papillary thyroid carcinoma.

Cancer Cytopathology | May 2017 | Vol. 125(5): 323-331

Liena Zhao, MD, PhD; Dora Dias-Santagata, PhD; Peter M. Sadow, MD, PhD; and William C. Faquin, MD, PhD



BACKGROUND: The noninvasive follicular variant of papillary thyroid carcinoma (PTC) has an indolent clinical behavior in comparison with other PTCs, including the invasive follicular variant of papillary thyroid carcinoma (IFVPTC). Recently, the term noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was introduced to emphasize the low biological potential of these tumors. This study compares clinical, cytological, and molecular features of NIFTP and IFVPTC.

METHODS: The study consisted of 97 thyroid fine-needle aspiration biopsy (FNAB) cases with corresponding surgical pathology from the pathology archives of the Massachusetts General Hospital. The collected patient data included the following: age, sex, type of surgery, tumor size, and prior cytological diagnosis with The Bethesda System for Reporting Thyroid Cytopathology. A molecular analysis using anchored multiplex polymerase chain reaction was per- formed for all cases. Each case was reviewed and subclassified histologically as NIFTP or IFVPTC. Cytology slides were scored semiquantitatively for nuclear atypia. The statistical analysis was performed with the nonparametric Mann-Whitney test.

RESULTS: The 97-case cohort consisted of 50 NIFTP cases and 47 IFVPTC cases, including 18 encapsulated IFVPTC cases and 29 nonencapsulated IFVPTC cases. Differences in the type of surgery (P5.0399), molecular features (P 5.0141), cytological classification (P 5.0266), and nuclear scores (P 5.0141) between NIFTP and IFVPTC were observed. There was overlap in the cytological classification of NIFTP and IFVPTC; however, NIFTP was more often classified as atypia of undetermined significance/follicular lesion of undetermined significance or follicular neoplasm/suspicious for fol- licular neoplasm in comparison with both subsets of IFVPTC. NIFTP was primarily associated with mutations in RAS, whereas an equal number of IFVPTC cases were associated with BRAFV600E or with RAS mutations.

CONCLUSIONS: Despite differences in the cytological classification and molecular profiles between NIFTP and IFVPTC, the degree of overlap makes it unlikely that most cases of NIFTP and IFVPTC can be accurately distinguished with FNAB.


Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst.
Genes, Chromosomes and Cancer

Genes, Chromosomes and Cancer | April 2017 | Vol. 56(4): 266–277

Natalya V. Guseva, Omar Jaber, Munir R. Tanas, Aaron A. Stence, Ramakrishna Sompallae, Jenna Schade, Allison N. Fillman, Benjamin J. Miller, Aaron D. Bossler, and Deqin Ma


Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis.


Primary Bone Anaplastic Large Cell Lymphoma Masquerading as Ewing Sarcoma: Diagnosis by Anchored Multiplex PCR.
Journal of Pediatric Hematology/Oncology

Journal of Pediatric Hematology/Oncology | April 2017

Susan Swee-Shan Hue, MBBS, PhD, FRCPA, Prasad Iyer, MBBS, MD, FRCPCH, Luke Han Wei Toh, MBBS, FRCR, MMed, Sudhanshi Jain, BSc, Enrica Ee Kar Tan, MBBS, MRCPCH, Kesavan Sittampalam, MBBS, FRCPA, Derrick Wen Quan Lian, MBBS, FRCPath, and Kenneth Tou En Chang, MBChB, FRCPath


A 3-year-old boy presented with pathologic fracture of the left proximal femur. Magnetic resonance imaging revealed an aggressive expansile bony mass associated with cortical destruction and surrounding myositis. Computed tomography–guided biopsy revealed a monomorphic small round blue cell tumor by histology. CD99 immunoreactivity and low-level EWSR1 gene translocation by break-apart fluorescent in situ hybridization initially favored a diagnosis of Ewing sarcoma and chemotherapy commenced. Subsequent molecular evaluation by an anchored multiplex poly- merase chain reaction–based assay (Archer FusionPlex Sarcoma Panel) revealed a nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) gene fusion. The diagnosis was then amended to primary bone ALK-positive anaplastic large cell lymphoma and the chemotherapy regimen was modified accordingly. This report illustrates the value of this molecular assay in establishing the correct diagnosis of a very rare malignancy masquerading as another tumor type.


Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.

Nature Medicine | May 2017

Ahmet Zehir, Ryma Benayed, Ronak H Shah, Aijazuddin Syed, Sumit Middha, Hyunjae R Kim, Preethi Srinivasan, Jianjiong Gao, Debyani Chakravarty, Sean M Devlin, Matthew D Hellmann, David A Barron, Alison M Schram, Meera Hameed, Snjezana Dogan, Dara S Ross, Jaclyn F Hechtman, Deborah F DeLair, JinJuan Yao, Diana L Mandelker, Donavan T Cheng, Raghu Chandramohan, Abhinita S Mohanty, Ryan N Ptashkin, Gowtham Jayakumaran, et al.



Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.


Detection of Novel t(12;17)(p12;p13) in Relapsed Refractory Acute Myeloid Leukemia by Anchored Multiplex PCR(AMP)-based Next-Generation Sequencing.

Applied Immunohistochemistry & Molecular Morphology | February 2017

Talha Badar, MD, Laura Johnson, PhD, Katelyn Trifilo, BS, Helen Wang, PhD, Brian A. Kudlow, PhD, Eric Padron, MD, Peter R. Pappenhausen, PhD, and Mohammad O. Hussaini, MD



Although several technologies can be used to detect gene fusions, anchored multiplex PCR next-generation se- quencing (AMP-NGS) offers the advantage of novel fusion de- tection and the ability to multiplex multitudinous genes. We applied AMP-NGS technology in the evaluation of a 56-year- old gentleman with myelodysplastic syndrome transformed acute myeloid leukemia (AML). Patient was initially diagnosed with low-risk myelodysplastic syndrome-refractory cytopenias and multilineage dysplasia (MDS-RCMD), progressed to AML after failing hypomethylating agent therapy. At progression patients had normal cytogenetics but NGS profiling showed ETV6 c.416_417del CT frame shift and U2AF1 S34F mutations. Patient attains brief remission of 2 months after induction chemotherapy and then he was refractory to 2 salvage chemo- therapy regimens. Reassessment after failing second salvage, identified t(12;17)(p13;p13)[20] by karyotype. It was postulated that the 12p13 locus might represent a new rearrangement of ETV6. AMP-NGS confirmed involvement of the ETV6 with discovery of a novel fusion partner, HIC1. The detection of the novel fusion partners was supported by the breakpoints origi- nally observed by karyotype. This discovery of ETV6-HIC1 gene fusion by AMP-NGS technology provided new insight into a leukemogenic pathway in AML. Future use of this technology can serve as an adjunct tool in workup of patients with AML and can also help in formulating therapeutic strategies.


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

Journal of Clinical Pathology | January 2017 | Vol. 70(8): 697-701

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.


MET Exon 14 Skipping Mutation in Non-Small Cell Lung Cancer Identified by Anchored Multiplex PCR and Next- Generation Sequencing.
Journal of Cancer Epidemiology and Prevention

Journal of Cancer Epidemiology and Prevention | December 2016

Tafe LJ, de Abreu FB, Peterson JD and Tsongalis GJ


Recurrent MET exon 14 (MET ex14) somatic splice site mutations have been described in 0.6 to 7% of lung non-small cell carcinomas (NSCLC) [1-3]. These mutations result in exon 14 skipping and subsequent MET activation with clinical trials demonstrating promising sensitivity to c-MET inhibitors [3]. These mutations are typically mutually exclusive of other lung adenocarcinoma known oncogenic driver mutations (e.g., EGFR, KRAS, BRAF, ERBB2, ALK, ROS1) but frequently co-occur with MDM2 and CDK4 amplification on chromosome 12q [2]. MET ex 14 mutations display diversity with upwards of 126 distinct DNA sequence variants described necessitating comprehensive genomic profiling by clinical laboratories for routine detection of these mutations in patients [2]. Of note, these MET ex14 splice site mutations are not limited to just NSCLC but also have been identified in a small percentage of gliomas and gastro-esophageal carcinomas [2,4]... continued in article


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.


MET Exon 14 Skipping in Non-Small Cell Lung Cancer.
The Oncologist

The Oncologist | April 2016 | Vol. 21(4): 481-486

Rebecca S. Heist, Hyo Sup Shim, Shalini Gingipally, Mari Mino-Kenudson, Long Le, Justin F. Gainor, Zongli Zheng, Martin Aryee, Junfeng Xia, Peilin Jia, Hailing Jin, Zhongming Zhao, William Pao, Jeffrey A. Engelman and A. John Iafrate



Background: Non-small cell lung cancers (NSCLCs) harboring specific genetic alterations can be highly sensitive to targeted therapies.

Materials and Methods: We performed a targeted rearrange- ment assay on 54 NSCLCs across all stages that were from patients who were never smokers and did not have driver mutations. Because MET exon 14 skipping was the most frequent alteration found, we surveyed the results for MET exon 14 skipping at Massachusetts General Hospital (MGH) since the inclusion of this alteration into our current molecular profiling panel.

Results: In a cohort of 54 never-smokers with lung cancers that were wild-type for known driver mutations, MET exon 14 skipping was the most frequently recurring alteration, occurring in 10 cancers (19%). Clinical testing at MGH via our next-generation sequencing (NGS) and NGS-rearrangement panels showed an additional 16 cases of MET exon 14 skipping, for an overall estimated frequency of 5.6%. A clinical case of a patient with MET exon 14 skipping treated with the MET inhibitor crizotinib is also described.

Conclusion: MET exon 14 skipping is a targetable gene alteration found in NSCLC. Patients with these alterations may respond well to MET inhibition.


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

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



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.


Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts.

Gastrointestinal Endoscopy | 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



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.


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 Ye



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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