Amplification Of Fusion Gene Research Articles

Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania.

Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.

Diagnostic utility of one-stop fusion gene panel to detect TFE3/TFEB gene rearrangement and amplification in renal cell carcinomas

MiT family translocation renal cell carcinoma (MiT-RCC) harbors translocations involving the TFE3 or TFEB genes. RCC with TFEB amplification is also identified and is associated with a more aggressive clinical course. Accurate diagnosis of MiT-RCC is crucial for patient management. In this study, we evaluated the performance of the Archer FusionPlex assay for detection of MiT-RCC with TFE3 or TFEB translocations and TFEB amplifications. RNA was extracted from 49 RCC FFPE tissue samples with known TFE3/TFEB status (26 TFE3 FISH positive, 12 TFEB FISH positive, 4 TFEB amplified (1 case both split and amplified), and 8 FISH negative) using the Covaris extraction kit. Target enriched cDNA libraries were prepared using the Archer FusionPlex kit and sequenced on the Illumina NextSeq 550. We demonstrate that the age of the specimen, quality of RNA, and sequencing metrics are important for fusion detection. Fusions were identified in 20 of 21 cases less than 2 years old, and TFE3/TFEB rearrangements were detected in all cases with Fusion QC ≥ 100. The assay identified intrachromosomal inversions in two cases (TFE3-RBM10 and NONO-TFE3), usually difficult to identify by FISH assays. TFEB mRNA expression and the TFEB/TFE3 mRNA expression ratio were significantly higher in RCCs with TFEB fusion and TFEB gene amplification compared to tumors without TFEB fusion or amplification. A cutoff TFEB/TFE3 ratio of 0.5 resulted in 97.3% concordance to FISH results with no false negatives. Our study demonstrates that the FusionPlex assay successfully identifies TFE3 and TFEB fusions including intrachromosomal inversions. Age of the specimen and certain sequencing metrics are important for successful fusion detection. Furthermore, mRNA expression levels may be used for predicting cases harboring TFEB amplification, thereby streamlining testing. This assay enables accurate molecular detection of multiple subtypes of MiT-RCCs in a convenient workflow.

The diagnostic profile of rare acute myelomegakaryoblastic leukemia

Acute myelomagakaryocytic leukemia is a diagnostic and therapeutic challenge owing to its heterogeneity and overlapping features with other types of acute leukemia. In order to build a diagnostic profile, we analyzed the biological, clinical and hematologic characteristics of acute myelomagakaryocytic leukemia. We found that, in three patients diagnosed with acute myelomagakaryocytic leukemia, there were two types of leukemia cells. One type was myeloblastic with positive peroxidase (POX) stainig and the expression of antigens CD13 and CD33. The other type was megakaryoblastic with negative POX staining and the expression of antigens CD36, CD41, CD42a and CD61. Three patients displayed the same cytogenetic abnormality, a (9: 22) translocation. Among the three patients with RT-PCR, two patients displayed BCR-ABL fusion gene amplification and one patient showed a previously undescribed OTT-MAL fusion gene amplification.

Genomic characterization of a PPP1CB-ALK fusion with fusion gene amplification in a congenital glioblastoma

ALK (Anaplastic lymphoma kinase) fusion proteins are oncogenic and have been seen in various tumors. PPP1CB-ALK fusions are rare but have been reported in a few patients with low- or high-grade gliomas. However, little is known regarding the mechanism of fusion formation and genomic break points of this fusion. We performed genomic characterization of a PPP1CB-ALK fusion with fusion gene amplification in a congenital glioblastoma. The PPP1CB-ALK consists of exons 1–5 of PPP1CB and exons 20–29 of ALK. The genomic translocation breakpoints were determined by real-time quantitative PCR (RT-qPCR) and Sanger sequencing of genomic DNA. Next generation sequencing, RT-qPCR and fluorescence in situ hybridization analyses demonstrated PPP1CB-ALK amplification. Copy number analyses of genes between PPP1CB and ALK using RT-qPCR suggest that the PPP1CB-ALK is likely the result of local chromothripsis followed by episomal amplification. Transcriptome sequencing demonstrated high-level SOX2 expression and predicted WNT/β-catenin pathway activation, suggesting possible therapeutic approaches.

EWSR1-NFATC2 Translocation-associated Sarcoma Clinicopathologic Findings in a Rare Aggressive Primary Bone or Soft Tissue Tumor.

In recent years, a novel small round cell sarcoma harboring EWSR1-NFATC2 translocation with immunomorphologic overlap with Ewing sarcoma (ES), myoepithelial tumors, and extraskeletal myxoid chondrosarcoma has emerged. There has not been a case series devoted to describing its detailed clinicopathologic and immunohistochemical characteristics. Six sarcomas harboring EWSR1-NFATC2 fusion transcripts by reverse transcription polymerase chain reaction and amplification of the fusion gene by fluorescence in situ hybridization were identified. The patients were 5 adult men and 1 adult woman. Three were primary bone tumors of the radius and 3 were primary soft tissue tumors. Most tumors showed monomorphic round to epithelioid cells in anastomosing cords and abundant myxohyaline to collagenous extracellular matrix. Two tumors had large areas of a solid, matrix-poor histomorphology. All tumors stained for CD99 and NKX2.2; while EMA, dot-like cytokeratin, and focal WT-1 and SMA were present in some tumors. All but 1 tumor showed poor histologic and radiologic responses to neoadjuvant ES-specific chemotherapy. Local or distant recurrences happened in 4 cases. EWSR1-NFATC2 sarcoma is a novel translocation-associated sarcoma. It presents as either a primary bone or soft tissue tumor, usually exhibits distinctive histopathologic features, and has predilection for long bones of adult men. It consistently shows recurrent fusion gene amplification readily detectable by EWSR1 breakapart fluorescence in situ hybridization, which serves as a diagnostic surrogate. It has potential for local and distant recurrence and histologic progression, and is resistant to Ewing sarcoma-specific chemotherapy.

Genomic amplification of BCR-ABL1 fusion gene and its impact on the disease progression mechanism in patients with chronic myelogenous leukemia

Identification of BCR-ABL1 fusion gene amplification status is critically important in the effective management of chronic myelogenous leukemia (CML) patients. Earlier reports suggested that overexpression of BCR-ABL1 either through amplification of BCR-ABL1 fusion gene or by the up regulation of BCR-ABL1 transcript level might be an early phenomenon in the establishment of IM resistance and disease evolution in CML. In the current study, we performed dual color dual fusion locus specific BCR/ABL1 FISH analysis along with karyotype analysis using GTG banding (G-banding using trypsin and Giemsa) technique in 489 patients with different clinical stages of CML at diagnosis or during the course of the disease to unravel the spectrum of BCR-ABL1 fusion gene amplification status. Among the study group analyzed, it was found that prevalence of occurrence of BCR-ABL1 fusion gene amplification was significantly higher in advanced stages of disease and in IM resistant CML-CP patients when compared to initial stage of disease, de novo CML-CP. Cytogenetic and metaphase FISH characterization on our study samples revealed that BCR-ABL1 fusion gene amplification was occurred through the formation of extra copies Ph chromosomes and isoderived Ph chromosomes. Current study suggests that unrestrained activity of BCR-ABL1 played a vital role in resistance to targeted therapy and disease evolution in CML. In our study population, patients in progressive stage CML and in IM resistant CP with multiple copies of BCR-ABL1 fusion gene displayed a poor response to targeted treatment with IM. Hence, the early identification of BCR-ABL1 fusion gene amplification using FISH technique will lead to improved interventions and outcome in future CML patients.

Rapid response of brain metastasis to crizotinib in a patient with KLC1-ALK fusion and MET gene amplification positive non-small cell lung cancer: a case report.

Non-small cell lung cancer (NSCLC) ranks as the leading cause of cancer-related death in the world. Brain metastasis (BM) is a common complication of NSCLC, with 25%–40% of patients developing BM during the course of the disease. A significant strategy of local disease control in the central nervous system is radiation therapy. With the development of precision medicine, the concept of treating lung cancer BM has gradually changed. In this case, we performed a surgical procedure to obtain enough tumor tissue for the detection of the target gene and other related experiments after the patient was informed. Finally, we found that the patient had both hepatocyte growth factor receptor (MET) gene amplification and kinesin light chain 1-anaplastic lymphoma kinase fusion (KLC1-ALK) through next-generation sequencing and showed sensitivity to the targeted therapy of crizotinib. The patient exhibited good response. Our case was successful and underwent targeted therapy with the guidance of precise diagnosis.

Therapeutic effects of crizotinib in lung cancer and the treatment after drug resistance

Crizotinib is a tyrosine kinase inhibitor (TKI), which is a target for echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK). It can prolong the progression free survival (PFS) of ALK positive patients with advanced non-small cell lung cancer (NSCLC). The median PFS in the first-line and second-line mPFS is 10.9 months and 7.7 months. However, despite an initial benefit, patients inevitably experience tumor progression, due to the ALK fusion gene amplification and secondary mutations of ALK kinase domain. Clinical trials show the promising efficacy like next generation ALK inhibitors and heat shock protein 90 (HSP90) can overcome acquired resistance. Key words: Lung neplasms; Drug resistance, neoplasm; Therapy

Abstract PR03: Novel ceritinib resistance mechanisms: new resistant mutation, fibroblast growth factor receptor 3 overexpression and cMET amplification-mediated ceritinib resistance

Abstract Anaplastic lymphoma kinase (ALK) rearrangement, which was first identified in non-small cell lung cancer (NSCLC) in 2007, is observed in 3%-5% of non-small-cell lung cancer (NSCLC) patients. For the treatment of ALK-rearranged non-small-cell lung cancer (NSCLC) patient, crizotinib, cMET/ALK/ROS1-TKI was tested and has shown remarkable tumor shrinkage in clinical trials for the treatment of advanced ALK-rearranged NSCLC patients. However, these tumors inevitably develop resistance to crizotinib due to various secondary mutations, ALK fusion gene amplification, or bypass pathway activation mediated by other receptor tyrosine kinases. To overcome crizotinib resistance, multiple ALK-TKIs have been developed. Ceritinib, a next-generation ALK-TKI, showed great efficacy in crizotinib-refractory, ALK-rearranged NSCLC patients in a clinical trial. However, the emergence of resistance to ALK-TKI is a major limitation for the effective therapy. Although various secondary mutations or bypass pathway activation-mediated resistance have been identified, large part of the resistance mechanism is still unknown. In this study, we explored ceritinib-resistant mechanisms using ceritinib-resistant, echinoderm microtubule-associated protein-like 4-ALK-positive H3122 cells and ceritinib-resistant patient-derived cells. Genetic alteration was examined by sequencing of cancer related genes. Protein expression related to ceritinib was examined by receptor-tyrosine kinase (RTK) array, immunoblotting and IHC. To overcome the identified resistance, single or combination treatment of the kinase inhibitors was testes in vitro and in vivo. We identified three novel ceritinib resistance mechanisms: a resistance mutation (L1198F), fibroblast growth factor receptor 3 (FGFR3) activation, and cMET gene amplification. L1198F-mutated ALK was the most sensitive to crizotinib. Molecular dynamic structure simulation successfully predicted the affinity of ceritinib or crizotinib to wild type and L1198F-mutated ALK. The patient-derived cMET-amplified ceritinib-resistant cells were sensitive to crizotinib, and the FGFR3-upregulated, ceritinib-resistant H3122 cells were re-sensitized to ALK-TKIs by FGFR inhibitor co-treatment. Our study revealed multiple ceritinib resistance mechanisms and suggest that some of the ceritinib resistance can be overcome by crizotinib. Citation Format: Ryohei Katayama, Takuya Sakashita, Noriko Yanagitani, Kengo Takeuchi, Makoto Nishio, Naoya Fujita. Novel ceritinib resistance mechanisms: new resistant mutation, fibroblast growth factor receptor 3 overexpression and cMET amplification-mediated ceritinib resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR03.

O10.1 - Next generation ALK inhibitors and mechanisms of resistance to therapy

ABSTRACT Anaplastic lymphoma kinase (ALK) gene rearrangements are important therapeutic targets in non-small cell lung cancer (NSCLC) that impart sensitivity to treatment with ALK tyrosine kinase inhibitors (TKIs), such as crizotinib. Indeed, crizotinib is now considered a standard of care for ALK-positive NSCLC patients based upon randomized trials in which crizotinib produced significant improvements in objective response rate (ORR) and progression-free survival (PFS) compared to first- and second-line cytotoxic chemotherapy. Nonetheless, patients almost invariably relapse on crizotinib—typically within 1-2 years. Molecular mechanisms of resistance to crizotinib include: 1) acquired secondary mutations in the ALK kinase domain (e.g., L1196M, G1269A), 2) ALK fusion gene amplification, and 3) activation of bypass signaling pathways, such as EGFR and c-KIT. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and/or structurally distinct, next-generation ALK inhibitors. Ceritinib (LDK378) is one such next-generation ALK inhibitor that recently gained accelerated regulatory approval in the United States. In a recent phase I study, ceritinib was associated with an ORR of 58% among 114 patients receiving at least 400 mg per day. Importantly, among patients previously treated with crizotinib, the response rate to ceritinib was 56%. Similarly, the next-generation ALK inhibitor alectinib has shown impressive activity in ALK-positive NSCLC. In a phase I/II study conducted in Japan, alectinib was associated with an ORR of 93.5% in TKI-naive patients. More recently, data on alectinib in the crizotinib-resistant setting has also emerged. Among 44 patients previously treated with crizotinib, the ORR on alectinib was 55%. Of note, both alectinib and ceritinib have also shown activity against central nervous system metastases—an important sanctuary site in ALK-positive NSCLC. Finally, at least 6 other ALK inhibitors are currently in clinical development. Determining the mechanisms of resistance to these agents as well as the optimal sequencing of ALK inhibitors in the clinic are important emerging questions.

Multilevel Molecular Profiling to Dissect Resistance to Tyrosine Kinase Inhibitors in TEL/ABL Positive Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemias (ALLs) bearing TEL/ABL (ETV6/ABL1) fusion gene are generally rare but can be identified within the Ph-like (BCR/ABL-like) ALLs - a poor risk ALL subtype characterized by the presence of various molecular lesions activating kinase signaling. Identification of these druggable lesions brought the possibility to improve the outcome of Ph-like ALL by incorporation of targeted therapeuticals – e.g. tyrosine kinase inhibitors (TKI) – into the current treatment strategies. In vitro studies proved the efficiency of TKI to inhibit TEL/ABL kinase activity justifying TKI use in clinical practice. However, there is a lack of knowledge on potential development of TKI resistance and its mechanism in TEL/ABL-positive ALL.To address this issue we aimed to establish TKI resistant TEL/ABL-positive cell line and study the mechanism of acquired resistance. After a long-term exposure to gradually growing doses of imatinib mesylate we induced a stable pan-TKI resistance not resulting from the most common mechanisms described in BCR/ABL ALL such as ABL kinase domain mutations, fusion gene amplification or enhanced fusion gene expression/activity.To elucidate the molecular basis of the resistance we performed multilevel molecular profiling. Using high density whole genome SNP array we identified a single acquired copy number aberration in the resistant cell line - a 60 kb intragenic deletion in KDM6A gene encoding lysine-specific histone demethylase. However, this deletion did not result in expression of the predicted aberrant protein. Using whole exome sequencing we identified 25 non-synonymous single nucleotide variants (SNVs) within gene coding regions to be gained by the resistant cell line. Only half of these variants (12/25) affected genes transcribed in the studied cell line and were thus subjected to further verification by Sanger sequencing. Finally, 5 of the 12 SNVs were confirmed to be stably present and expressed in the resistant cell line. All the affected genes – AEBP1, GNB1, KMT2C, RREB1, STAM2 – are involved in important biological processes and have not been associated with TKI resistance yet.Gene expression profiling of multiple replicates of parental sensitive and induced resistant cell line identified a gene expression pattern associated with TKI resistance consisting of more than 500 genes with significantly changed expression. Further analysis of this pattern revealed broad changes in many unrelated biological pathways and processes, hence providing a limited potential to specifically focus the study direction.We further employed two independent methods to study the resistant cell line proteome.Using 2-dimensional electrophoresis of the whole cell protein lysates we detected a single differentially expressed protein spot present in the resistant compared to parental sensitive cell line. Strikingly, mass spectrometry identified this protein as a transducin beta chain, product of the aforementioned GNB1 gene, one of the five beta subunits of human G-proteins playing an important role as signal transmitters.Using Size-Exclusion Chromatography-Microsphere-based Affinity Proteomic array (SEC-MAP; Wu, MCP 2009) we analyzed the total expression of more than 300 proteins by flow cytometry. We have found 39 proteins to be differentially expressed in resistant compared to parental sensitive cell line. Interestingly, several distinctive changes indicate enhanced B-cell receptor signaling in the resistant cell line. This mechanism was already implicated as a potential strategy of resistant cells for evading TKI induced apoptosis in BCR/ABL-positive leukemia (Klein, Cell Cycle 2004).In conclusion we have successfully used several high-throughput technologies to study genomic, transcriptomic and proteomic changes associated with acquired TKI resistance in TEL/ABL-positive ALL model and identified dysregulation of G-protein and BCR-signaling as its potential causes.Support: IGA MZ NT/13170-4; GAUK 694414; GAUK 596912; UNCE 204012; RVO-FNM64203; BIOCEV –(CZ.1.05/1.1.00/02.0109) from the ERDF DisclosuresNo relevant conflicts of interest to declare.

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Concomitant occurrence of EGFR (epidermal growth factor receptor) and KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations in an ALK (anaplastic lymphoma kinase)-positive lung adenocarcinoma patient with acquired resistance to crizotinib: a case report

BackgroundAnaplastic lymphoma kinase-positive non-small cell lung carcinoma patients are generally highly responsive to the dual anaplastic lymphoma kinase and MET tyrosine kinase inhibitor crizotinib. However, they eventually acquire resistance to this drug, preventing the anaplastic lymphoma kinase inhibitors from having a prolonged beneficial effect. The molecular mechanisms responsible for crizotinib resistance are beginning to emerge, e.g., in some anaplastic lymphoma kinase-positive non-small cell lung carcinomas the development of secondary mutations in this gene has been described. However, the events behind crizotinib-resistance currently remain largely uncharacterized. Thus, we report on an anaplastic lymphoma kinase-positive non-small cell lung carcinoma patient with concomitant occurrence of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations upon development of crizotinib-resistance.Case presentationA 61-year-old Caucasian never-smoking male was diagnosed with anaplastic lymphoma kinase -positive pulmonary adenocarcinoma, stage T4N3M1b. Treatment with crizotinib initially resulted in complete objective response in the thorax and partial response in the abdomen, but after 8 months of therapy the patient acquired resistance and progressed. Biopsies from new metastases revealed development of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations concomitant with the original anaplastic lymphoma kinase gene rearrangement and without signs of anaplastic lymphoma kinase fusion gene amplification or secondary anaplastic lymphoma kinase mutations.ConclusionTo our knowledge, this is the first report of an anaplastic lymphoma kinase-positive pulmonary adenocarcinoma, which upon emergence of crizotinib resistance acquired 2 new somatic mutations in the epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes, respectively, concomitant with the original anaplastic lymphoma kinase rearrangement. Thus, these 3 driver mutations, usually considered mutually exclusive, may coexist in advanced non-small cell lung carcinoma that becomes resistant to crizotinib, presumably because heterogeneous tumor clones utilize epidermal growth factor receptor and/or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog signaling to circumvent the inhibition of anaplastic lymphoma kinase-mediated signaling by crizotinib. The identification of new targetable somatic mutations by tumor re-biopsy may help clarify the mechanism behind the development of the acquired crizotinib resistance and pave the way for combined strategies involving multiple targeted therapies.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective

BackgroundNewcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking.Material and methodsTo ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30–80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis.ResultsThe deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province.ConclusionsTaken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

Abstract 37: Multiplexed mutational profiling of Japanese lung adenocarcinoma patients for personalized cancer therapy.

Abstract Background: Integration of genotype-based stratification into the lung cancer clinic is essential to implement the personalized medicine for patients with lung adenocarcinoma. Thus we have developed a multiplexed tumor genotyping panel for detecting somatic mutations relevant to molecular targeted therapies for lung cancer. The mutation profile data are communicated to clinicians to assign patients to appropriate therapy and/or clinical trials. Methods: Multiplexed tumor genotyping panel was developed to assess 23 mutations in 9 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN and HER2), 5 genes amplifications (EGFR, MET, PIK3CA, FGFR1 and FGFR2), and 5 fusion genes (EML4-ALK, CD74-ROS1, SLC34A2-ROS1, KIF5B-RET and CCDC6-RET) using pyrosequencing plus capillary electrophoresis, qRT-PCR, and multiplex RT-PCR, respectively. A written informed consent was obtained upfront from all patients enrolled into this study. Surgically resected tissues, tumor biopsies, pleural effusions and/or pericardial effusions from 280 patients were collected between July 2011 and July 2012 and used for tumor genotyping. Results: Somatic mutations were detected in 52.9% (148/280) of all patients. The most common mutation detected in this study was EGFR mutation (35.4%, 99) followed by KRAS mutation (10.4%, 29). EGFR L858R (52.5%) and KRAS G12C (44.8%) was the most frequent among EGFR and KRAS mutations, respectively. Other detected mutations were PIK3CA mutation (2.5%), PIK3CA amplification (2.5%), EGFR amplification (2.5%), MET amplification (2.1%), EML4-ALK fusion gene (1.4%), BRAF mutation (1.1%), NRAS mutation (1.1%), HER2 mutation (1.1%), MEK1 mutation (0.7%), AKT1 mutation (0.7%), KIF5B-RET fusion gene (0.4%), CD74-ROS1 fusion gene (0.4%) and FGFR1 amplification (0.4%). EGFR tyrosine kinase inhibitor (EGFR-TKI) was administered to 71.9% (41/57) of previously untreated patients who harbor EGFR activating mutations. Among those treated with EGFR-TKIs, simultaneous mutations in other genes with EGFR mutations were detected in 21.9% (9/41). Response rate of EGFR-TKIs in patients with simultaneous mutations (44.4%) was lower than that in patients with EGFR mutation alone (68.8%), suggesting that additional mutations may play some role in evading oncogenic signaling blockade. EGFR mutations were detected less frequently in smokers than in never-smokers (25.1% vs 60.5%, p<0.0001). Meanwhile, KRAS mutations were detected more frequently in smokers than in never-smokers (13.6% vs 2.5%, p=0.0043), which is consistent with previous reports. The majority of patients with never-smoking history harbored targetable somatic mutations compared to those with smoking history (71.6% vs 45.2%, p<0.0001). Conclusions: We have established the multiplexed tumor genotyping panel and this should be incorporated into lung cancer clinical practice to facilitate personalized cancer medicine. Citation Format: Masakuni Serizawa, Yasuhiro Koh, Hirotsugu Kenmotsu, Mitsuhiro Isaka, Akane Naruoka, Junko Suzuki, Masaru Watanabe, Masahiro Endo, Takashi Nakajima, Shoji Takahashi, Yasuhisa Ode, Tateaki Naito, Tetsuhiko Taira, Akira Ono, Toshiaki Takahashi, Nobuyuki Yamamoto. Multiplexed mutational profiling of Japanese lung adenocarcinoma patients for personalized cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2013-37

Acute myeloid leukemia with promyelocytic morphology lacking RARA rearrangement and with double minutes, MYC deletion and 2 cell lines with amplification of MYC region: case report and literature review

Acute promyelocytic leukemia (APL) typically demonstrates the translocation t(15;17)(q24;q21) leading to PML-RARA fusion in ~92 % of the cases. The remaining t(15;17) negative APL-like cases may have other cytogenomic rearrangements, including cryptic PML-RARA fusion, translocation variants, or MYC gene amplification. The morphologic and cytogenetic profile of the APL-like cases with MYC amplification and the prognostic implications are largely unknown due to rarity of these cases. Here, we present a case of acute myeloid leukemia with promyelocytic morphology lacking the classic t(15;17) and RARA rearrangement, with der(5)t(5;17)(q13;21), MYC deletion, double minutes, and two cell lines with amplification of the MYC region. The two cell lines differed in contribution to the proportion of the cells and copy number of the amplified probes during the clinical course. This is likely the first case with such a complex molecular cytogenetic evolution detailed in the same patient. Literature review revealed that most of the APL-like cases with MYC amplification have no t(15;17) and PML-RARA (78.5 %; 7/8 cases) but a complex karyotype (75 %; 6/8 cases). Deletion 17p/monosomy 17 and deletion 9p/monosomy 9 are reported in ~40–50 % of the cases and are associated with poor prognosis. MYC amplification alone may not be a reliable prognostic indicator in APL-like cases. Our findings can be very helpful for clinicopathologic/cytogenetic correlation and potentially management of such rare but potentially unrecognized APL-like patients.

Genomic and clinical analysis of fusion gene amplification in rhabdomyosarcoma: A report from the Children's Oncology Group

Alveolar rhabdomyosarcoma (RMS) is an aggressive pediatric cancer of the myogenic lineage with frequent chromosomal translocations involving the PAX3 or PAX7 and FOXO1 genes. Based on previous studies indicating that the fusion genes are amplified in a subset of these cancers, we conducted a comprehensive molecular and clinical investigation of these amplification events. Using oligonucleotide arrays to localize amplicons, we found that the minimal 1p36 amplicon measured 0.13 Mb and only contained PAX7 whereas the minimal 13q14 amplicon measured 0.53 Mb and contained FOXO1 and the poorly characterized LOC646982 gene. Application of a fluorescence in situ hybridization assay to over 100 fusion-positive cases revealed that the fusion gene is amplified in 93% of PAX7-FOXO1-positive and 9% of PAX3-FOXO1-positive cases. While most cells in amplified PAX7-FOXO1-positive cases contained the amplicon, only a fraction of cells in the amplified PAX3-FOXO1-positive cases contained the amplicon. Expression studies demonstrated that the fusion transcripts were generally expressed at higher levels in amplified cases, and that the PAX7-FOXO1 fusion transcript was expressed at higher levels than the PAX3-FOXO1 fusion transcript. Finally, fusion gene amplification and PAX7-FOXO1 fusion status were each associated with significantly improved outcome; a multivariate analysis demonstrated that this predictive value was independent of other standard prognostic parameters. These findings therefore provide further evidence for a novel good prognosis subset of fusion-positive RMS.

Oncogene Amplification as an Incidental Finding in FISH Testing for Gene Rearrangements in Lymphoid Hematopoietic Neoplasms

Abstract 2505Oncogene amplification resulting in overexpression, although common in solid tumors, is rare in hematopoietic neoplasms. This is particularly true in lymphoid neoplasms compared to AML where MYC, MLL or RUNX1 (AML1) amplification has been mostly seen, and to CML where BCR/ABL fusion gene amplification has been also reported. Typically, lymphoid neoplasms are tested at diagnosis by FISH for specific reciprocal chromosome translocations that lead to overexpression of deregulated oncogenes such as BCL1, BCL2, BCL6 and MYC in B-cell lymphoma and myeloma or BCR/ABL gene fusion in ALL. Nevertheless, we have unexpectedly seen oncogene amplification from time to time in our FISH lab. To study the incidence of gene amplification and its diagnostic and clinical implications, we retrospectively analyzed FISH results routinely performed on paraffin embedded lymphoma tissues, lymph nodes, bone marrow aspirates and peripheral bloods in the past three and half years using translocation probes for BCL2/IGH, BCL1/IGH, MYC/IGH and BCR/ABL and break apart probes for BCL6 and MYC. The highest amplification rate seen was in BCL2/IGH testing: 11 of the 1,710 cases were positive (0.643%). In 2 cases with follicular lymphoma (FL), BCL2 amplification presented as homogenously staining regions (hsr), one case with diffuse large B-cell lymphoma (DLBCL) showed double minutes (dmin), and two cases with FL had a pattern of combined hsr and dmin. Five cases with low grade FL intriguingly showed a similar pattern of BCL2/IGH translocation and concomitant amplification of the rearranged BCL2 as a small hsr. The remaining case diagnosed as Burkitt lymphoma (BL) was positive for MYC/IGH translocation and for BCL2 amplification present as large hsr. BCL6 amplification was observed in 4 of 1,537 cases tested (0.26%). In all these cases, amplification presented as hsr. One case diagnosed as EBV+ DLBLC was positive for MYC/IGH translocation and 3’ BCL6 high level amplification. Another case with FL showed BCL6 rearrangement and 5’ BCL6 amplification. BCL1 amplification with translocation was seen as hsr in 1 case with mantle cell lymphoma out of 2,898 tested. BCL1 amplification was observed in another case with plasma cell myeloma as hsr out of 3,413 tests performed. MYC gene amplification was positive in 3 of 2,186 (0.137%). One case with BL showed MYC rearrangement with a concomitant 3’ deletion and 5’ amplification. The second case diagnosed as B-cell lymphoma with features intermediate between DLBCL and BL showed highly amplified MYC gene as hsr. The third case was DLBCL with 15–50 copies of MYC gene per cell as dmin. Among 530 BCR/ABL tests ordered for acute leukemia or ALL, 2 (0.377%) cases with T cell lymphoblastic leukemia/lymphoma (T-ALL/LBL) showed ABL amplification as episomes (4–8 copies in one case; 6–30 in another). Both were abnormal by cytogenetics analysis, but negative for t(9;22)(q34;q11.2) and without dmin or hsr identified. Although detection rates of oncogene amplification seem to be very low with limited specific translocation probes applied to lymphoid neoplasms, they may well be higher when FISH signal patterns are analyzed more carefully. In our study, 5 of the 11 FL cases tested for IGH/BCL2 showed a peculiar pattern positive for the t(14;18)-IGH/BCL2 and a concomitant red signal amplification of BCL2, the size of which is usually small and may be overlooked under microscope. All these 5 cases had low grade FL, suggesting that oncogene amplification can be an early genomic event in lymphomagenesis. MYC gene amplification is generally considered a late stage genomic alteration. When MYC is rearranged, amplification of BCL2, BCL6 or BCL1 may need to be taken into consideration for disease stratification, or vice versa. For instance, so called “double-hit” or “triple-hit” lymphomas currently require two or three concurrent translocations for diagnosis. Oncogene amplification, equally important for deregulation/overexpression as a translocation, should be considered as a second or a third hit in diagnosis and differential diagnosis of B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL. FISH is a very useful tool to identify episomal oncogene amplification. Episomes, unlike cytogenetically evident dmin and hrs, are invisible by chromosome analysis, but their detection is important as reported for integration of targeted tyrosine kinase inhibitors into chemotherapeutic regimens. Disclosures:No relevant conflicts of interest to declare.

Identification of a new translocation that disrupts the RUNX1 gene in a patient with de novo acute myeloid leukemia

Translocation (8;21)(q22;q22)/RUNX1-RUNX1T1 is a molecular marker that is usually associated with a favorable outcome in both pediatric and adult patients with acute myeloid leukemia (AML). The present report describes the results of hematologic, cytogenetic, and fluorescence in situ hybridization analysis of a case of AML with maturation in a 23-year-old woman. Cytogenetic analysis revealed a balanced translocation involving chromosomal band 21q22, which disrupts the RUNX1 gene, and 10q22, with the following karyotype: 45,X,-X,t(10;21)(q24;q22)[cp16]/46,XX [4]. Interphase FISH showed, in 67% of the 300 interphase nuclei analyzed, three signals for RUNX1 and two RUNX1T1, but no signals corresponding to RUNX1-RUNX1T1 fusion gene. These results were corroborated by RT-PCR, which revealed negative results for the amplification of RUNX1-RUNX1T1 fusion gene. The patient was refractory to conventional and salvage chemotherapy regimens and early relapsed after unrelated donor bone marrow transplantation (BMT), dying of pneumonia, acute respiratory failure, and sepsis on day +80 after BMT, 1 year after diagnosis.

Identification of Bcl-2/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis

The determination of polymerase chain reaction (PCR) amplification product sizes of the Bcl-2/IgH fusion gene from follicular lymphoma (FL) provides evidence of clonal identity. The present study describes detection of Bcl-2/IgH fusion gene clonality utilizing a small, simple microcapillary electrophoretic chip combined with a real-time PCR method. The microcapillary electrophoretic chip system effectively detects size differences among the Bcl-2/IgH fusion gene amplification products of FL from patient samples; something that is not possible using traditional gel electrophoresis. We also describe the potential of this system to utilize formalin-fixed, paraffin-embedded tissue samples sectioned on charged slides. The simple detection of Bcl-2/IgH fusion gene clonality using a microcapillary electrophoretic chip provides reliable information for monitoring minimal residual disease of FL, and can be an effective tool for use in clinical laboratories.