RET Gene Fusions Research Articles

PL01.01 Tumor-Agnostic Biologically Driven Treatments: An Endless Dream?

PL01.01 Tumor-Agnostic Biologically Driven Treatments: An Endless Dream?

EP1.14-03 Driver Genes as Predictive Indicators of Brain Metastasis in Patients with Advanced NSCLC: EGFR and ALK as Well as RET Gene Mutations

Brain metastasis is a cause of disease progression and death in lung cancer patients, and is also one of the most common metastatic sites of lung cancer. Non-small lung cancer (NSCLC) accounts for about 80% of all lung cancer patients, and adenocarcinoma has become the main subtype of NSCLC in recent years. A retrospective analysis verified the role of gene mutations in brain metastasis in patients with non-small lung cancer (NSCLC). The clinicopathological data of 552 patients who received driver genes detection for lung cancer in the Affiliated Cancer Hospital of Zhengzhou University/Henan Cancer Hospital from January 2015 to June 2017 were collected; NGS was used for gene detection. The driving genes for detection were EGFR, ALK, KRAS, ROS-1, BRAF, ERBB2, RET and c-MET, which were eight lung cancer driving genes recommended in NCCN guidelines. All the tests were carried out in the Center of Molecular Pathology in the Affiliated Tumor Hospital of Zhengzhou University. Of the 552 patients with advanced NSCLC, 153 (27.7%) had brain metastases. Univariate analysis showed that age (P = 0.008), gender (P = 0.016), smoking history (P = 0.010), lymph node metastasis (P = 0.003), and three driver genes: positive EGFR mutation (P = 0.001), positive ALK gene fusion (P = 0.021) and positive RET gene fusion (P = 0.003) were factors influencing the incidence of brain metastasis. Logistic multivariate regression analysis revealed that only positive EGFR mutation (P = 0.012), positive ALK gene fusion (P = 0.015), positive RET gene fusion (P = 0.003), pathological type (P = 0.009), lymph node N2–3 metastasis (P = 0.000) and a younger age (P = 0.000) were independent risk factors for brain metastasis. In addition, a ROC curve was plotted with the above factors with AUC=0.705 (P=0.000). EGFR mutation, ALK gene fusion and RET gene fusion in advanced NSCLC patients play roles in brain metastasis as positive driver genes.

Spindle Cell Tumors With RET Gene Fusions Exhibit a Morphologic Spectrum Akin to Tumors With NTRK Gene Fusions

A major breakthrough in the classification of soft tissue tumors has been the recent identification of NTRK-fusion related neoplasms which are amenable to highly effective targeted therapies. Despite these therapeutic opportunities, diagnostic challenges have emerged in recognizing tumors characterized by protein kinase fusions, as they are associated with a wide morphologic spectrum, variable risk of malignancy and a rather nonspecific immunoprofile. As such, NTRK-related fusions may occur in infantile fibrosarcoma, lipofibromatosis-like neural tumors (LPF-NTs), tumors resembling malignant peripheral nerve sheath tumors, etc. Triggered by an index case resembling LPF-NT but harboring RET gene rearrangement, we investigated our files for cases showing RET gene abnormalities to establish their clinicopathologic features. Tumors were tested with a combination of targeted RNA sequencing and fluorescence in situ hybridization methods. Six cases with RET gene rearrangements were identified, all except 1 occurred in children, including 4 infants. Their morphologic spectrum was quite diverse, but closely reproduced the phenotype of NTRK-fusion-positive tumors, including LPF-NTs (n=3), infantile fibrosarcoma-like tumor (n=2) and malignant peripheral nerve sheath tumor-like (n=1). Three cases showed coexpression of S100 and CD34, whereas the remaining 3 had a nonspecific immunoprofile. The tumors ranged morphologically and clinically from benign to highly malignant. None of the LPF-NT cases recurred, whereas 2 patients with malignant histology had a highly aggressive course with distant metastases to lung and other viscera. By targeted RNA sequencing these tumors harbored RET fusions with an identical break in exon 12, which retains the tyrosine kinase domain in the fusion oncoprotein and involving various gene partners (CLIP2, CCDC6, SPECC1L, MYH10, and NCOA4). Our results suggest that RET fusion-positive neoplasms share a similar phenotypic spectrum with the NTRK-positive tumors, displaying either fibroblastic or neural-like differentiation, and spanning a wide spectrum of clinical behavior. These findings open new avenues for targeted therapy with RET inhibitors currently available in clinical trials.

Analysis of Cell-Free DNA from 32,989 Advanced Cancers Reveals Novel Co-occurring Activating RET Alterations and Oncogenic Signaling Pathway Aberrations.

RET is an emerging oncogenic target showing promise in phase I/II clinical trials. An understudied aspect of RET-driven cancers is the extent to which co-occurring genomic alterations exist and how they may impact prognosis or therapeutic response. Somatic activating RET alterations were identified among 32,989 consecutive patients with metastatic solid tumors tested with a clinical cell-free circulating tumor DNA (cfDNA) assay. This comprehensive next-generation sequencing (NGS) assay evaluates single-nucleotide variants, and select indels, fusions, and copy number gains in 68-73 clinically relevant cancer genes. A total of 176 somatic activating RET alterations were detected in 170 patients (143 fusions and 33 missense mutations). Patients had non-small cell lung (NSCLC, n = 125), colorectal (n = 15), breast (n = 8), thyroid (n = 8), or other (n = 14) cancers. Alterations in other oncogenic signaling pathway genes were frequently identified in RET-positive samples and varied by specific RET fusion gene partner. RET fusions involving partners other than KIF5B were enriched for alterations in MAPK pathway genes and other bona fide oncogenic drivers of NSCLC, particularly EGFR. Molecular and clinical data revealed that these variants emerged later in the genomic evolution of the tumor as mechanisms of resistance to EGFR tyrosine kinase inhibitors. In the largest cancer cohort with somatic activating RET alterations, we describe novel co-occurrences of oncogenic signaling pathway aberrations. We find that KIF5B-RET fusions are highly specific for NSCLC. In our study, only non-KIF5B-RET fusions contributed to anti-EGFR therapy resistance. Knowledge of specific RET fusion gene partner may have clinical significance.

Sensitivity of next-generation sequencing assays detecting oncogenic fusions in plasma cell-free DNA

ObjectivesPlasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility. Materials and methods: Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360™(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling. ResultsOf 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360. ConclusionFocusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.

First experience of LOXO-292 in the management of pediatric patients with RET-altered cancers.

10045 Background: Genomic alterations of RET kinase, including chromosomal fusions and activating point mutations, have been identified as oncogenic drivers in pediatric and adult cancers. Multikinase inhibitors have modest activity against RET-altered cancers and are associated with significant toxicity. LOXO-292 is a potent, ATP-competitive, small molecule RET inhibitor with high selectivity for RET, preclinical activity in the brain and excellent pharmacokinetic properties. In an ongoing phase 1/2 study of adult and adolescent patients with advanced RET-altered cancers, LOXO-292 demonstrated marked antitumor activity and was well tolerated. Methods: Pediatric patients with RET-altered cancers who were unable to access LOXO-292 through a phase 1/2 clinical trial were enrolled using FDA-allowed, IRB-approved single patient protocols . Patients received LOXO-292 (capsule/liquid formulation) orally, continuously, at a starting dose of 90 mg/m2 BID. Response was assessed locally by investigators. Results: As of January 31, 2019, 4 female patients aged 13 months–8 years were enrolled from the Republic of Korea (papillary thyroid cancer, n = 1) and the USA (infantile myofibroma/hemangiopericytoma, n = 1; congenital mesoblastic nephroma/infantile fibrosarcoma, n = 1; lipofibromatosis, n = 1). All tumors harbored RET gene fusions (5′ partners: CCDC6, MYH10, SPECC1L, and NCOA4, respectively), detected by next-generation sequencing. Previous therapies included surgery/iodine 131 (n = 1); vandetanib (n = 1); surgery/chemotherapy (n = 1); 1 patient was treatment-naïve. Duration of treatment ranged from 3–120 days and all patients remain on treatment. There were no dose modifications or discontinuations due to adverse events. There were no treatment-related adverse events ≥ grade 3. Two patients who were evaluable for response had partial responses (both ongoing, 1 confirmed, 1 pending confirmation). Conclusions: These preliminary data from a real-world setting suggest that LOXO-292 is effective and safe in pediatric patients whose tumors harbor RET gene fusions.

Performance of Oncomine Fusion Transcript kit for formalin-fixed, paraffin-embedded lung cancer specimens.

Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for precision medicine. We used formalin‐fixed, paraffin‐embedded tissue samples of non‐small cell lung cancer from 150 EGFR mutation‐negative cases and 10 fusion status‐known cases and compared the performance of the Oncomine Dx Fusion Transcript Test (ODxFT) with FISH break‐apart for the detection of ALK, RET, and ROS1 fusion genes. RNA was extracted from the paraffin‐embedded tissue samples with or without macrodissection under hematoxylin and eosin staining, and the ALK fusion gene was independently determined using these assays. Fusion detection analyses were successfully carried out using ODxFT in 150 cases, with only one invalid case. ALK fusion genes were detected at a frequency of 7.3% (11/150) in the lung cancer specimens. Concordance rate between the ODxFT and ALK‐FISH analyses was 99.3% (148/149). Sensitivity and specificity were 91.7% and 99.3%, respectively. All the samples with a known fusion status were accurately matched between the two assays. Our results show a high concordance rate between the ODxFT and ALK‐FISH analyses. ODxFT was thus validated as an effective method for detecting clinically significant ALK fusion genes in paraffin‐embedded tissue samples.

Genomic alterations and survival in young patients aged under 40 years with completely resected non-small cell lung cancer.

Young patients are rarely diagnosed with non-small cell lung cancer (NSCLC), and little is known about its predisposing genomic alterations and survival. This retrospective study was conducted to evaluate the genomic alterations, treatment and prognosis of young patients under 40 years old from a cohort of 640 lung adenocarcinoma and squamous carcinoma cases from Zhejiang Cancer Hospital. All patients were examined for EGFR, KRAS, NRAS, PIK3CA, BRAF and HER2 mutations and ALK, ROS1 and RET fusion genes. In total, 54 patients were aged under 40 years. The frequencies of genomic alterations in younger (≤40 years) and older (>40 years) patients were 68.5% and 54.8%, respectively (P=0.05). Younger patients harbored a higher frequency of fusion genes (22.2% vs. 4.1%, P<0.001) but not gene mutations (46.3% vs. 45.6%, P=0.92). There was a general trend toward shorter recurrence-free survival (RFS) (35.2 vs. 43.8 months, P=0.050), while no overall survival (OS) difference existed between younger and older patients (50.2 vs. 51.4 months, P=0.112). Younger patients with NSCLC had a higher frequency of gene fusions than older patients and had a trend of worse OS.

Analysis of the frequency of oncogenic driver mutations and correlation with clinicopathological characteristics in patients with lung adenocarcinoma from Northeastern Switzerland

BackgroundMolecular testing of lung adenocarcinoma for oncogenic driver mutations has become standard in pathology practice. The aim of the study was to analyze the EGFR, KRAS, ALK, RET, ROS1, BRAF, ERBB2, MET and PIK3CA mutational status in a representative cohort of Swiss patients with lung adenocarcinoma and to correlate the mutational status with clinicopathological patient characteristics.MethodsAll patients who underwent molecular testing of newly diagnosed lung adenocarcinoma during a 4-year period (2014–2018) were included. Molecular analyses were performed with Sanger sequencing (n = 158) and next generation sequencing (n = 311). ALK, ROS1 and RET fusion gene analyses were also performed with fluorescence in situ hybridization and immunohistochemistry/immunocytochemistry. Demographic and clinical data were obtained from the medical records.ResultsOf 469 patients with informative EGFR mutation analyses, 90 (19.2%) had EGFR mutations. KRAS mutations were present in 33.9% of the patients, while 6.0% of patients showed ALK rearrangement. BRAF, ERBB2, MET and PIK3CA mutations and ROS1 and RET rearrangements were found in 2.6%, 1.9%, 1.9%, 1.5%, 1.7% and 0.8% of the patients, respectively. EGFR mutation was significantly associated with female gender and never smoking status. ALK translocations were more frequent in never smokers, while KRAS mutations were more commonly found in ever smokers. The association between KRAS mutational status and female gender was statistically significant only on multivariate analysis after adjusting for smoking.ConclusionThe EGFR mutation rate in the current study is among the higher previously reported mutation rates, while the frequencies of KRAS, BRAF, ERBB2 and PIK3CA mutations and ALK, ROS1 and RET rearrangements are similar to the results of previous reports. EGFR and KRAS mutations were significantly associated with gender and smoking. ALK rearrangements showed a significant association with smoking status alone.

Expanding the Molecular Characterization of Thoracic Inflammatory Myofibroblastic Tumors beyond ALK Gene Rearrangements

IntroductionHalf of inflammatory myofibroblastic tumors (IMTs) regardless of anatomic location harbor anaplastic lymphoma kinase gene (ALK) rearrangements and overexpress anaplastic lymphoma kinase protein. The wide application of next-generation sequencing and the clinical benefit to tyrosine kinase inhibitors have opened new opportunities for investigation of ALK-negative IMTs. MethodsIn this study, we have investigated a series of pediatric and adult thoracic IMTs for abnormalities in a wide spectrum of actionable kinases by applying a variety of molecular and next-generation sequencing techniques, including fluorescence in situ hybridization (FISH), targeted RNA sequencing, and NanoString assay. ResultsThere were 33 patients with thoracic IMTs, including five children; their mean age was 37. The tumors showed a monomorphic spindle cell phenotype, except for one with an epithelioid morphologic pattern and moderate to severe atypia. By immunohistochemistry, 24 tumors were ALK positive, and 19 of the 24 showed ALK rearrangements and one ret proto-oncogene gene (RET) rearrangement by FISH. RNA sequencing was performed in the remaining four cases lacking ALK abnormalities by FISH, revealing ALK fusions involving tropomyosin 4 gene (TMP4) and echinoderm microtubule associated protein like 4 gene (EML4) as partner in three cases. NanoString assay was performed in the remaining case, revealing ALK alternative transcription initiation (ALKATI). Nine cases lacking ALK abnormalities were further tested by FISH or targeted RNA sequencing, revealing ROS1 rearrangement in six cases and ETS variant 6 gene (ETV6)–neurotrophic receptor tyrosine kinase 3 gene (NTRK3) fusion in three cases, respectively. ConclusionsBy using a battery of complementary molecular techniques, we have shown that all the thoracic IMTs harbored a tyrosine kinase abnormality, with 30% involving a kinase gene other than ALK, including ROS1, NTRK3, and RET gene fusions. We have also described for the first time ALKATI-induced ALK oncogenic activation in IMTs.

A Case of CCDC6-RET Fusion Mutation in Adult Acute Lymphoblastic Leukemia (ALL), a Known Activating Mutation Unreported in ALL

INTRODUCTIONUnderstanding the oncogenesis and the role of the different mutations observed are of crucial importance. They not only have prognostic importance, but also are evolving therapeutic targets. The first targeted mutation in ALL was the BCR-ABL gene(Thomas et al. 2004; Kim et al. 2015), which not only improved overall response and survival in “fit” patients, but also allowed for a promising alternative treatment strategy for elderly or frail patients(Delannoy et al. 2006). More recently, a subgroup of patient were noticed to share the same gene expression profile as Ph-positive, but lack the BCR-ABL translocation. These patients have several different genomic alteration, and are possibly targeted using different TKI's(Den Boer et al. 2009).CASE REPORTA 55 year-old female patient with past medical history of type 2 diabetes and hypothyroidism presented to the ED with 2 weeks of right flank pain radiating to her right groin. Her initial vital signs were stable, and she was afebrile. Her physical exam was remarkable for petechiae with no adenopathy or organomegaly.The patient's initial blood work was remarkable for pancytopenia with normal, coagulation studies, uric acid, renal and liver function except for elevated Alkaline phosphatase (481 U/L) Lactate dehydrogenase (17216 U/L). A bone marrow flow cytometry revealed a population of blasts that represented 77% of the sample. The blast were positive for CD9, CD19, CD10 (variable), CD20, cCD22, CD34, CD45, cCD79a, HLA-DR, and cTdT. These findings were compatible with B-lymphoblastic leukemia. Cytogenetics display abnormal 71-74, del(22) with high hypertriploidy. FoundatioOneHeme® was sent as part of her diagnostic work up, and revealed CCDC6-RET Fusion, FLT3 D835H and D835V, CDKN2A/B loss, DNMT3A Truncation in intron 14, MLL3 S793* and TP53 K132R as genomic alterations.The patient achieved complete response after 1 cycle of R-HyperCVAD and finished induction with this regimen. Afterwards, patient underwent myeloablative allogenic bone marrow transplant from. The patient developed Graft versus host disease 4 months after transplant that required steroids and Ruxolitinib. To date, 413 days since transplant, she continues to be in remission.DISCUSSIONActivating mutations involving tyrosine kinase receptors and other molecules involved in intracellular signal amplifications/transduction are known to be important drivers of oncogenesis. Moreover, the presence of the same mutation across different neoplasm, often represents an important pharmacological target. CCDC6-RET and other common fusion partners leads to homodimerization causing ligand independent activation(Truebestein and Leonard 2018). RET autophosphorylation lead to activation of several intracellular transduction cascades involved in cellular proliferation, including MAPK, AKT, PKC, JAK-STAT, PKA and PI3K(Santoro and Carlomagno 2013).RET fusion genes were first seen in patients with PTC, and then identified in subset of patient with NSCLC(Wang et al. 2012). These mutations are considered to have a considerable role in oncogenesis as they are usually mutually exclusive with other alterations such as BRAF and RAS in PTC(Xu et al. 2017; Mitsutake et al. 2006; Kimura et al. 2003), a hypothesis that is supported by clinical response to inhibition of such tyrosine kinase receptors.Several multikinase inhibitors with RET inhibition are available and have been approved for several different indications. Several TKI commercially available have been used in trials of NSCLC and other clinical settings and represent a potential option. RET TKI has showed worse clinical response when compared to EGFR, BRAF and ALK inhibition, possibly due to off target effect leading to poor compliance or less active RET inhhibition. Newer RET specific molecules currently being developed and evaluated in different clinical scenarios(Drilon et al. 2017). To our knowledge, there is no data regarding CCDC6-RET targeting in hematological malignancies.CONCLUSIONThis is the first report of CCDC6-RET rearrangement in ALL to date. The isolation of CCDC6-RET mutation raise the possibility of directed RET inhibition with selective or multitarget TKI in this disease. Comprehensive assessment of RET rearrangement and point mutations should be conducted in ALL in order to identify the prevalence and clinical implications of this new finding. DisclosuresNo relevant conflicts of interest to declare.

Detection of RET rearrangements in papillary thyroid carcinoma using RT-PCR and FISH techniques - A molecular and clinical analysis

IntroductionOncogenic BRAF and RAS mutations as well as multiple known (and yet unknown) RET fusion oncogenes comprise the majority of causative molecular alterations in papillary thyroid carcinoma (PTC). Apparently “mutation-negative” PTCs encompass a heterogenous group impeding analysis of prognostic significance of underlying genetics. Material and methodsBRAF wild type PTC tissue of 56 patients was analyzed using two established methods: hybrid-specific RT-PCR for the predominant rearrangement RET/PTC1 and fluorescent in situ hybridization (FISH). Clinical features of the cases with and without RET rearrangement were compared (patient age, gender, tumor size, focality, lymph node affection, and iodine avidity). ResultsRT-PCR revealed RET/PTC1 rearrangements in five of 56 tumors (9%). FISH confirmed these, and identified four additional RET rearrangements (9/56; 16%). Loss of the iodine avidity only occurred in cases of RET/PTC hybrids (7/9 tumors), but not in RET/PTC-negative PTCs (0/41 tumors with available uptake information; p = 0.029). The risk to develop lymph node metastases was eight times higher in presence of RET rearrangements (p = 0.010). ConclusionsFISH analysis, in contrast to hybrid-specific RT-PCR, revealed infrequent and unknown RET fusion genes. The presence of RET rearrangements was associated with a significantly elevated risk to develop iodine refractory disease and lymph node metastases. Of note, significant clinical discrimination was only achievable when taking the FISH results into account; differences would have been missed when using the RT-PCR method only. Increasing evidence of the clinical impact of RET/PTC-positivity may influence the decision on the extent of surgical resection, especially on lymph node dissection, in PTCs.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study

BackgroundGene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material.MethodsWe describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples.ResultsReproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies.ConclusionThis methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Abstract 645: Targeted next generation sequencing in molecular diagnostics of solid tumors

Abstract Cancer genomics role in patients management underlines the importance of rigorous validation of molecular diagnostics approaches to guide treatment decisions. Nowadays only next generation sequencing (NGS) assays can satisfy the need to match a comprehensive tumor molecular profile to the right therapies. Nevertheless, given the complexity, cost, and outcome implications for patients, a clinically valid NGS assay requires careful consideration of gene panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, performance with low quality/quantity of nucleic acids, and workflow feasibility. In this study we describe the introduction of the Oncomine Comprehensive Assay (OCA) into a Molecular Pathology diagnostic framework. The OCA analyzes 143 genes of which 73 oncogenes are interrogated for mutational hotspots and 26 tumor suppressor genes for all exons. In addition, the OCA detects copy number variations in 49 genes and fusion drivers in 22 genes. Multiplexed PCR-based DNA and RNA libraries were sequenced on Ion S5 and data analysis was performed by the Torrent Suite using the Oncomine pipeline. The OCA was clinically validated analyzing a cohort of 200 tumor samples of 4 solid tumor types (100 Non Small Cell Lung Cancers, 40 Colon Adenocarcinomas, 40 Pancreatic Adenocarcinomas, 20 Esophageal Adenocarcinomas), for which routine molecular analysis was available. We successfully identified relevant somatic point mutations, indels, and high-level CNAs: TP53, KRAS and EGFR were the genes most frequently mutated though alteration frequencies varied among different tumor types, as expected. OCA DNA profiling demonstrated 95% concordance with standard methods for detecting commonly targeted somatic variants with highly concordant observed variant allele frequencies for detection of KRAS, EGFR and BRAF mutations. Also high-level CNAs strongly correlated with available routine standard tests. The concordance of OCA analysis and standard tests at the RNA level was definitely lower (80%): a subset of 45 lung samples were tested for ALK, RET, ROS1 rearrangements by OCA RNA assay and we successfully detected 15 of the 24 expected fusions, indicating the feasibility of identifying them by this NGS panel, but also stressing the limits of a targeted approach to detect widely variable breakpoints in the analyzed gene fusions. In order to detect all the ALK, ROS1, and RET fusions variants, we perform the parallel validation of the Archer FusionPlex RNA-based ALK, RET, and ROS1 gene fusion assay in 8 lung adenocarcinomas. Results are under evaluation. We conclude that the OCA DNA analysis can be a valuable tool in solid tumor molecular diagnostics being able to identify both common and additional relevant variants beyond current routine practice, and represents a broadly applicable targeted NGS assay which allows advanced precision oncology for different solid tumors types. Citation Format: Greta Grassini, Valeria De Pascali, Ilaria Francaviglia, Gilda Magliacane, Elena Dal Cin, Anna Talarico, Chiara Iacona, Claudio Doglioni, Lorenza Pecciarini, Maria Giulia Cangi. Targeted next generation sequencing in molecular diagnostics of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 645.

Biobanking and the further development of precision medicine

Over the last 15years, an estimated 3000 large centralized biobanks have been established worldwide, making important contributions to the further development of precision medicine. In many cases, these biobanks are affiliated with pathological institutes or work closely with them. In which translational research projects, and during which phases in the development of new drugs are human bioprobes being used and can their use be easily traced in the literature? PubMed, Internet research, and information from the German Biobank Alliance and the European initiative BBMRI-ERIC. High-quality biosamples from centralized biobanks are increasingly used in clinical research and development projects. Success stories, where bioprobes have contributed to the further development of precision medicine, are shown in this paper using among others the example of RET gene fusion discovery in lung cancer. Interestingly enough, many key publications in the field of precision medicine do not contain exact references to the biobanks involved. The importance of centralized biobanks in translational research and clinical development is constantly increasing. However, in order to ensure the acceptance and visibility of biobanks, their participation in success stories of biomedical progress must be systematically documented and published.

Genetic landscape of papillary thyroid carcinoma in the Chinese population.

Improvement in the clinical outcome of human cancers requires characterization of the genetic alterations underlying their pathogenesis. Large-scale genomic and transcriptomic characterization of papillary thyroid carcinomas (PTCs) in Western populations has revealed multiple oncogenic drivers which are essential for understanding pathogenic mechanisms of this disease, while, so far, the genetic landscape in Chinese patients with PTC remains uncharacterized. Here, we conducted a large-scale genetic analysis of PTCs from patients in China to determine the mutational landscape of this cancer. By performing targeted DNA amplicon and targeted RNA deep-sequencing, we elucidated the landscape of somatic genetic alterations in 355 Chinese patients with PTC. A total of 88.7% of PTCs were found to harbor at least one candidate oncogenic driver genetic alteration. Among them, around 72.4% of the cases carried BRAF mutations; 2.8% of cases harbored RAS mutations; and 13.8% of cases were characterized with in-frame gene fusions, including seven newly identified kinase gene fusions. TERT promoter mutations were likely to occur in a sub-clonal manner in our PTC cohort. The prevalence of somatic genetic alterations in PTC was significantly different between our Chinese cohort and TCGA datasets for American patients. Additionally, combined analyses of genetic alterations and clinicopathologic features demonstrated that kinase gene fusion was associated with younger age at diagnosis, larger tumor size, and lymph node metastasis in PTC. With the analyses of DNA rearrangement sites of RET gene fusions in PTC, signatures of chromosome translocations related to RET fusion events were also depicted. Collectively, our results provide fundamental insight into the pathogenesis of PTC in the Chinese population. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical and Translational Implications of RET Rearrangements in Non-Small Cell Lung Cancer.

Since the discovery in 2012 of rearranged during transfection proto-oncogene gene (RET) rearrangements in NSCLC, at least 12 different fusion variants have been identified, with kinesin family member 5B gene (KIF5B)-RET being the most frequent and the best characterized. Unlike ALK receptor tyrosine kinase gene (ALK) and ROS1 rearrangements, RET fusion genes cannot be adequately detected by immunohistochemistry (IHC), although fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction are fully complementary diagnostic tools. In large retrospective studies, RET rearrangements correlate with adenocarcinoma histologic subtype, never-smoking status, younger age, more advanced disease stage, potentially higher chemosensitivity (in particular, to pemetrexed-based regimens), and coexistence of other genomic alterations. To date, several preclinical models, clinical trials, and retrospective studies have investigated multitarget inhibitors with anti-rearranged during transfection proto-oncogene (RET) activity in patients with RET-rearranged lung cancer. In the clinical setting, the benefit in terms of response (16%-47%) and progression-free survival (2-7 months) is clearly not comparable to that seen with other targeted agents in oncogene-addicted NSCLC. Furthermore, multikinase agents showed high rates of severe toxicities, leading to frequent dose reduction and drug discontinuation. To date, no definitive conclusions about a potentially different impact of anti-RET therapies according to RET fusion variants have been drawn on account of discordant data coming mostly from small subgroup analyses. Importantly, the absence of a striking clinical benefit in RET oncogene-addicted NSCLC underscores theclear need for development of more selective andpotentRET inhibitors and for better characterization of concomitant genomic alterations and mechanisms of resistance to RET inhibition in patients with lung cancer.

MS 25.05 Novel Fusions

Chromosomal rearrangements or deletions can generate novel gene fusions that lead to the expression of chimeric proteins with oncogenic activity in lung and other cancers. The paradigm for gene fusions in lung cancer is the EML4-ALK gene fusion which is found in approximately 5% of lung adenocarcinomas.1 Once identified, drug development for this target proceeded rapidly and crizotinib was the first approved tyrosine kinase inhibitor for patients with ALK+ non-small cell lung cancer based on its ability to generate substation objective response rates and prolonged progression free survival (NSCLC).3,4 The development of CNS penetrant and/or next generation ROS1 inhibitors including lorlatinib, entrectinib and TPX-0005 is ongoing. RET gene fusions are also found in 1-2% of NSCLC, but the use of multiple different RET inhibitors have failed to reproduce the success of targeting ALK or ROS1 fusions.5 The development of more selective RET inhibitors such as LOXO-292, BLU-667, and RXDX-105 are currently in clinical trials for RET+ lung and other cancers. NTRK1 fusions were recently identified in NSCLC and homologous NTRK2 and NTRK3 fusions are found in multiple tumor types.6,7, Early clinical trials of larotrectinib showed an impressive objective response rate of 76% for 12 different tumor histologies harboring NTRK1/2/3 fusions and the CNS-penetrant entrectinib is similarly being evaluated in a basket trial. Other rare, novel fusions have recently been identified in NSCLC including EGFR fusions or MET fusions.7 Early evidence suggests that these fusions may also respond to cognate TKIs. EGFR fusions break the paradigm of ALK and ROS1 fusions in which the 5’ end of ALK or ROS1 is replaced with the 5’ portion of another gene. EGFR fusions retain most of the EGFR gene, with the unrelated gene sequencing fusing at the 3’ end of EGFR. A related oncogenic EGFR mutation in which the kinase domain of EGFR is duplicated in tandem has also been described and appears responsive to EGFR TKIs.7 Additional fusions involving the receptor tyrosine kinase (RTK) encoding genes AXL, PDGFRA, and ERBB4 fusions have been described, but little is known about the true incidence in lung cancer.8,9 The fusions described thus far all involve genes that encode RTKs, but additional gene fusions have also been identified in lung cancer including BRAF fusions.7,9 BRAF fusions replace the 5’ region of BRAF, including the Ras-binding domain (RBD), with sequences from another gene. Anecdotal evidence suggests that these alterations may be responsive to MEK inhibition. Analogous splice alteration which remove a region including the RBD have also been described in cancer and are oncogenic. Finally, gene fusions involving NRG1, which encodes the HER3/4 ligand neuregulin-1, have been described, mostly in invasive mucinous adenocarcinomas of the lung.9,10 Several challenges exist to the development of targeted therapies for these novel fusions. Even in the era of next-generation sequencing tests these alterations may go undetected due to limited testing of genes to those with approved therapies (ALK, ROS1, EGFR, and BRAF) and not all assays are designed to detect all of these alterations. Furthermore, the rarity of some of these alterations may make clinical trials for these novel fusions less appealing, although amalgamating some of these alterations with analogous mutations, e.g., MET gene fusions with MET exon 14 splice alterations or MET gene amplification, may allow for a more rapid path to approval.

419O - Safety of alectinib in non-small cell lung cancer patients with RET fusion gene (ALL-RET): Results from the dose-finding portion of a phase 1/2 study

419O - Safety of alectinib in non-small cell lung cancer patients with RET fusion gene (ALL-RET): Results from the dose-finding portion of a phase 1/2 study

PUB042 The Detection and Analysis of RET Gene Fusion in Non-Small Lung Cancer in High-Throughput Sequencing

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