Receptor Tyrosine Kinase Fusion Research Articles

Pilocytic astrocytoma: The paradigmatic entity in low‑grade gliomas (Review).

Among low-grade gliomas, representing 10-20% of all primary brain tumours, the paradigmatic entity is constituted by pilocytic astrocytoma (PA), considered a grade 1 tumour by the World Health Organization. Generally, this tumour requires surgical treatment with an infrequent progression towards malignant gliomas. The present review focuses on clinicopathological characteristics, and reports imaging, neurosurgical and molecular features using a multidisciplinary approach. Macroscopically, PA is a slow-growing soft grey tissue, characteristically presenting in association with a cyst and forming a small mural nodule, typically located in the cerebellum, but sometimes occurring in the spinal cord, basal ganglia or cerebral hemisphere. Microscopically, it may appear as densely fibrillated areas composed of elongated pilocytic cells with bipolar 'hairlike' processes or densely fibrillated areas composed of elongated pilocytic cells with Rosenthal fibres alternating with loosely fibrillated areas with a varied degree of myxoid component. A wide range of molecular alterations have been encountered in PA, mostly affecting the MAPK signalling pathway. In detail, the most frequent alteration is a rearrangement of the BRAF gene, although other alterations include neurofibromatosis type-1 mutations, BRAFV600E mutations, KRAS mutations, fibroblast growth factor receptor-1 mutations of fusions, neurotrophic receptor tyrosine kinase family receptor tyrosine kinase fusions and RAF1 gene fusions. The gold standard of PA treatment is surgical excision with complete margin resection, achieving minimal neurological damage. Conventional radiotherapy is not required; the more appropriate treatment appears to be serial follow-up. Chemotherapy should only be applied in younger children to avoid the risk of long-term growth and developmental issues associated with radiation. Finally, if PA recurs, a new surgical approach should be performed. At present, novel therapy involving agents targeting MAPK signalling pathway dysregulation is in development, defining BRAF and MEK inhibitors as target therapeutical agents.

KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway.

Here, we investigated the detailed molecular oncogenic mechanisms of a novel receptor tyrosine kinase (RTK) fusion, KLC1-ROS1, with an adapter molecule, KLC1, and an RTK, ROS1, discovered in pediatric glioma, and we explored a novel therapeutic target for glioma that possesses oncogenic RTK fusion. When wild-type ROS1 and KLC1-ROS1 fusions were stably expressed in the human glioma cell lines A172 and U343MG, immunoblotting revealed that KLC1-ROS1 fusion specifically activated the JAK2-STAT3 pathway, a major RTK downstream signaling pathway, when compared with wild-type ROS1. Immunoprecipitation of the fractionated cell lysates revealed a more abundant association of the KLC1-ROS1 fusion with JAK2 than that observed for wild-type ROS1 in the cytosolic fraction. A mutagenesis study of the KLC1-ROS1 fusion protein demonstrated the fundamental roles of both the KLC1 and ROS1 domains in the constitutive activation of KLC1-ROS1 fusion. Additionally, in vitro assays demonstrated that KLC1-ROS1 fusion upregulated cell proliferation, invasion, and chemoresistance when compared to wild-type ROS1. Combination treatment with the chemotherapeutic agent temozolomide and an inhibitor of ROS1, JAK2, or a downstream target of STAT3, demonstrated antitumor effects against KLC1-ROS1 fusion-expressing glioma cells. Our results demonstrate that KLC1-ROS1 fusion exerts oncogenic activity through serum-independent constitutive activation, resulting in specific activation of the JAK-STAT pathway. Our data suggested that molecules other than RTKs may serve as novel therapeutic targets for RTK fusion in gliomas.

Receptor tyrosine kinase- fusions in paediatric spindle cell tumors

Pediatric spindle cell tumors are rare and often difficult to diagnose due to asimilar morphology and anon-specific immunohistochemical profile. Genetic characterization of these lesions has been constantly improving, which has led to the identification of new subgroups that were partly included in the WHO classification. Receptor tyrosine kinase fusions play aspecial role in these tumors and their verification has diagnostic relevance and can be an option for target-oriented therapies. In the case of pediatric spindle cell tumors, genetic fusions form especially with NTRK1‑3, ALK, RET, and ROS1. Overall, pediatric tumors with receptor tyrosine kinase fusions are predominantly low-grade tumors, which are often subdivided into the group of intermediate-malign tumors.

A comprehensive survey of tumor mutation burden (TMB) using the AACR GENIE cbioportal database: Pathogenesis of NTRK and RET fusion (NTRK+/RET+) positive colorectal (CRC).

33 Background: Receptor tyrosine kinase fusion (RTK+) has been identified in colorectal carcinoma (CRC). Tumor agnostic treatment has been approved in NTRK fusion ( NTRK+) and RET fusion ( RET+) positive solid tumor. However, overall response rate (ORR) was the lowest among entrectinib-treated NTRK+ CRC (20%, 2/10) and selpercatinib treated RET+ CRC (20%, 2/10) among all NTRK+ and RET+ solid tumors respectively. Methods: We surveyed all 58 human RTKs to identify RTK fusions in CRC and RET and NTRK fusions in other solid tumors using AACR GENIE cbioportal public version 13.0. We abstracted and compared the tumor mutation burden (TMB) of these fusions from the cbioportal database. Patient and tumor characteristics were also abstracted. Results: Among the 14812 unique CRC patients identified in the AACR GENIE cbioportal, a total of 153 unique RTK fusions were identified (44 with known fusion partners, 109 with intragenic rearrangement) in 27 out of the total 58 human RTKs. The most common RTK fusions in CRC were in FGFR1 (N = 23), EGFR (N = 21), ERBB2, NTRK1, RET, FGFR2 (N = 10 each), FLT1 and FLT3 (N = 9 each), FLT4 and ALK (N = 7 each). Considering only samples with identified fusion partners, the mean TMB was 66.6 +/-15.8 (mt/MB) for NTRK+ CRC (N = 9), and 35 +/-11.5 for RET+ CRC (N = 4), which were significantly higher when compared to mean number of 6.2 +/- 5.4 of TMB for all other RTK+ CRC (ALK, EGFR, FGFR1/2, ERBB4, FLT1/3/4, LMKT2, MET, NTRK2, PDGFR-b, ROS1) (N = 30, P< 0.05). Furthermore, NTRK+ CRC harbored significantly higher TMB than RET+ CRC (P = 0.003). The mean TMB was 4.0 +/- 1.9 for RET+ NSCLC (N = 65), 2.6 +/- 1.6 for RET+ Thyroid cancer (N = 52), which were both significantly lower than RET+ CRC. Mean TMB was 3.1 +/- 1.7 for NTRK+ glioma (N = 21), 3.3 +/- 1.7 for NTRK+ thyroid cancer (N = 21), 6.5+/-5.1 for NTRK+ Melanoma (N = 8), 5.9 +/- 3.4 for NTRK+ NSCLC (N = 7), 5.1 +/- 1.2 for NTRK+ breast cancer (N = 7) all of them were significantly lower than the mean TMB of NTRK+ CRC. 1482 (10.0%) patients with CRC had their MSI status reported. 3 out of 3 NTRK+ CRC patients with known MSI status were all MSI-H (100%). 12 out of 12 non-NTRK/non-RET RTK+ CRC patients were MSI-S. Conclusions: In this comprehensive survey of human RTK fusions in CRC, NRTK+ and RET+ CRC harbor significantly higher TMB compared to other RTK+ CRC or to other NTRK+/RET+ solid tumors. Limited data showed NTRK+ CRC were all MSI-H. NTRK+ and RET+ CRC are distinct molecular subtypes of CRC with unique pathogenesis from all other NTRK+ or RET+ solid tumors. We hypothesize that NTRK+/RET+ CRC may benefit from pembrolizumab that has been approved for histologic agnostic high TMB tumors rather than RET or NTRK tyrosine kinase inhibitors (low ORR in CRC) that also have histological agnostic indications.

Oncogenic Drivers and Therapeutic Vulnerabilities in KRAS Wild-Type Pancreatic Cancer.

Approximately 8% to 10% of pancreatic ductal adenocarcinomas (PDAC) do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer is important to guide patient stratification for clinical trials of molecularly targeted agents. We analyzed a single-institution cohort of 795 exocrine pancreatic cancer cases (including 785 PDAC cases) with a targeted multigene sequencing panel and identified 73 patients (9.2%) with KRAS wild-type (WT) pancreatic cancer. Overall, 43.8% (32/73) of KRAS WT cases had evidence of an alternative driver of the MAPK pathway, including BRAF mutations and in-frame deletions and receptor tyrosine kinase fusions. Conversely, 56.2% of cases did not harbor a clear MAPK driver alteration, but 29.3% of these MAPK-negative KRAS WT cases (12/41) demonstrated activating alterations in other oncogenic drivers, such as GNAS, MYC, PIK3CA, and CTNNB1. We demonstrate potent efficacy of pan-RAF and MEK inhibition in patient-derived organoid models carrying BRAF in-frame deletions. Moreover, we demonstrate durable clinical benefit of targeted therapy in a patient harboring a KRAS WT tumor with a ROS1 fusion. Clinically, patients with KRAS WT tumors were significantly younger in age of onset (median age: 62.6 vs. 65.7 years; P = 0.037). SMAD4 mutations were associated with a particularly poor prognosis in KRAS WT cases. This study defines the genomic underpinnings of KRAS WT pancreatic cancer and highlights potential therapeutic avenues for future investigation in molecularly directed clinical trials. See related commentary by Kato et al., p. 4527.

METB-03. THE DISTINCT GENOMICS OF HIGH-GRADE INFANT GLIOMAS AMONG PEDIATRIC GLIOMAS HARBORING RECEPTOR TYROSINE KINASE FUSIONS

Abstract Gliomas account for about 60% of pediatric brain tumors, about half of which are classified as high-grade. Recent studies have shown that infants with high-grade gliomas (HGG) harboring receptor tyrosine kinase (RTK) fusions have a more favorable prognosis than pediatric HGG patients. The molecular mechanisms for this favorable, but from the histology divergent clinical outcome, are yet unknown. We analyzed n=18 brain tumor samples (n=7 infant, n=11 pediatric) of patients with low-grade glioma (LGGs) or HGG presenting with RTK fusions using single-cell RNA sequencing. Considering the different fusion genes (ALK n=6, NTRK n=8, and MET n=4), age groups, and glioma grades, we evaluated the metaprograms of tumor cells and their proportions using non-negative matrix factorization. Preliminary results show similar metaprograms comparing infant and pediatric LGG. Cells detected as MES-like did not occur in LGG but were found in several HGG samples. However, in HGG, the MES-like cells show different expression patterns for infant and pediatric samples. In addition, compared with pediatric HGG, infant HGG has a higher proportion of programs that are predominantly present in LGG, such as AC-like and NPC-like. These initial findings serve as a starting point for cell type-specific transcription factor regulatory networks and are an important step toward understanding the underlying biology of this distinct pediatric brain tumor entity.

METB-02. DECIPHERING THE CELLULAR HETEROGENEITY OF INFANT-TYPE HEMISPHERIC GLIOMAS USING SINGLE-NUCLEI MULTI-OMICS

Abstract Infant-type hemispheric gliomas (IHGs) have recently been recognized as a new tumor entity. Compared to other types of pediatric gliomas, most IHGs distinctively harbor a fusion in a cluster of receptor tyrosine kinase (RTK) genes: the NTRK family, ROS1, ALK, or MET. Clinically, IHG patients appear to be associated with better outcomes compared to older children with high-grade tumors. While large-scale genomic studies have helped uncovered the complex molecular landscape and clinical features of IHGs, little is known about the underlying biology – specifically how tumor heterogeneity may explain the spectrum of clinical presentations and progression of the disease. Using single-nuclei multi-omic (RNA/ATAC-seq) profiling, we assembled a comprehensive atlas of IHG tumor cells from a cohort of patients spanning different age groups and RTK fusion subtypes. We measured that tumor single-nuclei transcriptomes clustered into distinct subpopulations and contained genes expressed by the normal brain cell lineages. Data integration with single-cell atlases from the developing brain allowed us to rank tumor cell subpopulations according to their developmental stage, with age- and fusion-specific differences. Using trajectory inference and gene expression velocity modeling, we describe a hierarchical and dynamic organization between subpopulations, potentially illustrating the role of phenotypic plasticity in glioma formation. Moreover, we measured in fragments of open chromatin a cell-type specific enrichment of binding sites for transcription factors known to regulate brain cell maturation. Taken together, our multi-modal atlas describes the cellular and functional heterogeneity of IHGs, paving the way towards a greater understating of its unique biology and clinical consequences.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of immunotherapy in patients with high tumor mutational burden tumors

The development of novel antitumor agents and accompanying biomarkers has improved survival across several tumor types. Previously, we developed recommendations for tumor-agnostic treatments in patients with solid tumors with DNA mismatch repair deficient or neurotrophic receptor tyrosine kinase fusions. Recently, immune checkpoint inhibitors have shown efficacy in patient with tumor mutation burden-high (TMB-H) solid tumors and have been established as a third tumor-agnostic agent, making it necessary to develop the guideline prioritized for these patients. Clinical questions regarding medical care were formulated for patients with TMB-H advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO), Japanese Society of Medical Oncology (JSMO), and Japanese society of pediatric hematology/oncology (JSPHO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO, and the public comments among all societies' members was done. The current guideline describes three clinical questions and seven recommendations for whom, when, and how TMB should be tested, and what is recommended for patients with TMB-H advanced solid tumors. In this guideline, the committee proposed seven recommendations for performing TMB testing properly to select patients who are likely to benefit from immunotherapy.

PD-23 Comprehensive survey of AACR GENIE cBioPortal database of receptor tyrosine kinase fusion in colorectal carcinoma identified high co-occurring genomic alterations in NTRK1 & RET fusion-positive CRC

PD-23 Comprehensive survey of AACR GENIE cBioPortal database of receptor tyrosine kinase fusion in colorectal carcinoma identified high co-occurring genomic alterations in NTRK1 & RET fusion-positive CRC

Current perspectives on the management of patients with advanced RET-driven thyroid cancer in Europe.

The incidence of thyroid cancer is increasing worldwide with the disease burden in Europe second only to that in Asia. In the last several decades, molecular pathways central to the pathogenesis of thyroid cancer have revealed a spectrum of targetable kinases/kinase receptors and oncogenic drivers characteristic of each histologic subtype, such as differentiated thyroid cancer, including papillary, follicular, and medullary thyroid cancer. Oncogenic alterations identified include B-Raf proto-oncogene (BRAF) fusions and mutations, neurotrophic tyrosine receptor kinase (NTRK) gene fusions, and rearranged during transfection (RET) receptor tyrosine kinase fusion and mutations. Multikinase inhibitors (MKIs) targeting RET in addition to multiple other kinases, such as sorafenib, lenvatinib and cabozantinib, have shown favourable activity in advanced radioiodine-refractory differentiated thyroid cancer or RET-altered medullary thyroid cancer; however, the clinical utility of MKI RET inhibition is limited by off-target toxicity resulting in high rates of dose reduction and drug discontinuation. Newer and selective RET inhibitors, selpercatinib and pralsetinib, have demonstrated potent efficacy and favourable toxicity profiles in clinical trials in the treatment of RET-driven advanced thyroid cancer and are now a therapeutic option in some clinical settings. Importantly, the optimal benefits of available specific targeted treatments for advanced RET-driven thyroid cancer require genetic testing. Prior to the initiation of systemic therapy, and in treatment-naïve patients, RET inhibitors may be offered as first-line therapy if a RET alteration is found, supported by a multidisciplinary team approach.

Role of molecular testing for malignant pleural effusion in targeted therapy for advanced non‐small cell lung cancer

To confirm the predictive value of targeted therapies for oncogenic driver gene mutations detected in malignant pleural effusion (MPE) cell blocks from patients with advanced non-small cell lung cancer (NSCLC). For patients with NSCLC whose tumor tissues could not be used to detect oncogenic driver gene status, molecular mutation status in 101 MPE cell blocks was tested using amplification refractory mutation system polymerase chain reaction prior to treatment. Corresponding targeted therapies were adopted based on the detection results. Mutations observed in MPE cell blocks included epidermal growth factor receptor mutation (EGFR) (60.4% [61/101]), anaplastic lymphoma kinase fusion (6.3% [5/80]), and ROS proto-oncogene 1 receptor tyrosine kinase fusion (3% [2/70]). Other mutations that were found in <5% of patients included epidermal growth factor receptor-2, rat sarcoma-filtered germ carcinogenic homologous B1, neuroblastoma RAS viral oncogene homolog, and mesenchymal epithelial transition factor exon 14. The median follow-up time was 23.5 months for the 41 patients with a single EGFR mutation and who received tyrosine kinase inhibitor monotherapy as the first-line treatment; in these patients, the objective response rate was 78% (95% confidence intervals (CI), 62% to 89%), progression-free survival was 10.8 months (95% CI, 8.7 to 13.0 months), and overall survival was 31.7 months (95% CI, 13.9 to 49.4 months). Malignant pleural effusion cell blocks are recommended for mutation testing for targeted therapies in patients with NSCLC.

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.

Next-generation Sequencing as a Potential Diagnostic Adjunct in Distinguishing Between Desmoplastic Melanocytic Neoplasms.

Desmoplastic melanomas (DMs) are often challenging to diagnose and ancillary tests, such as immunohistochemistry, have limitations. One challenge is distinguishing DM from benign desmoplastic melanocytic neoplasms. In this study, we explored the utility of next-generation sequencing data in the diagnosis of DMs versus desmoplastic Spitz nevi (DSN) and desmoplastic nevi (DN). We sequenced 47 cases and retrieved 12 additional previously sequenced clinical cases from our dermatopathology database. The 59 total cases were comprised of 21 DMs, 25 DSN, and 13 DN. The DMs had the highest tumor mutation burden at 22 mutations/megabase (m/Mb) versus the DSN (6m/Mb) and DN (8m/Mb). Truncating mutations in NF1 resulting in a loss-of-function were exclusive to the DM cohort, identified in 8/21 (38%) cases. Importantly, missense mutations in NF1 were nonspecific and seen with similar frequency in the different cohorts. Other mutations exclusive to the DMs included truncating mutations in TP53 , CDKN2A , and ARID2 . Among the DSN, 17/25 (68%) had an HRAS mutation or receptor tyrosine kinase fusion consistent with other Spitz tumors. Two cases in the DN cohort had missense mutations in BRAF without additional progression mutations and 2 other cases had mutations in GNAQ , supporting a diagnosis of a sclerosing blue nevus. The remainder of the DN had nonspecific mutations in various signaling pathways with few progression mutations. Overall, our study provides preliminary data that next-generation sequencing data may have the potential to serve as an ancillary diagnostic tool to help differentiate malignant and benign desmoplastic melanocytic neoplasms.

PATH-14. MOLECULAR ANALYSIS OF ASTROCYTOMAS WITH A DOMINANT PILOMYXOID MORPHOLOGY

Abstract Pilomyxoid astrocytomas (PMAs) are tumors of infancy occurring in the hypothalamic chiasmatic region, with somewhat worse prognosis than pilocytic astrocytomas (PAs). We report molecular findings in PAs with a dominant pilomyxoid histomorphology across the various age groups and CNS locations. Foundation Medicine comprehensive genomic profiling (CGP) (FoundationOne, FoundationOneCDx, FoundationOneHeme) were performed during routine clinical care. A retrospective cohort analysis was done to evaluate the genomic characteristics of PMAs. 57 PMAs were identified, of which 6 occurred in patients &amp;lt; 12 months old (10.5%); 14 in 1-3 year old (24.6%); 15 in 4-10 y.o. (26.3%); 12 in 11-20 y.o. (21.1%), and 10 occurred in young adults 21-39 y.o. (17.5%). 30/57 (52.6%) were female and 27/57 (47.4%) male. 55 PMAs occurred in the brain and 2 in the spinal cord. Tumors were categorized by notable rearrangements and point mutations. The common fusion, KIAA1549:BRAF, was detected in 23/57 samples (40.4%). BRAF fusions with other gene partners (BRAF, TAX1BP1) occurred in 2, and BRAF V600E mutations in 4 patients. An additional 4 patients had FGFR1 fusions (FGFR1, TACC1) and 14 had FGFR mutations. Other alterations included 3 NF1 mutations, 2 NTRK family receptor tyrosine kinase fusions, 2 RAF gene fusions, 2 other (ATRX, ALK) alterations, and 1 VUS. Tumor Mutational Burden was under 10 mut/Mb for all patients. In this cohort of 57 PMAs, MAPK pathway genetic alterations were dominant drivers, similar to other PAs. We also observed other frequent genetic alterations not involving BRAF, including FGFR and NTRK gene family members that have FDA approved targeted inhibitors. While these also likely cause MAPK pathway activation, these non-BRAF variants are less common in PAs. Our report contributes to growing evidence that PMAs harbor a distinct molecular alteration profile, which is becoming increasingly important in the context of the evolving targeted therapeutics landscape.

An Unusual Cause of Acute Gastroenteritis, Ascites, and Eosinophilia in a 29-Year-Old Woman

An Unusual Cause of Acute Gastroenteritis, Ascites, and Eosinophilia in a 29-Year-Old Woman

Landscape of potentially targetable receptor tyrosine kinase fusions in diverse cancers by DNA-based profiling

Recurrent fusions of receptor tyrosine kinases (RTKs) are often driving events in tumorigenesis that carry important diagnostic value and are potentially targetable by the increasing number of tyrosine kinase inhibitors (TKIs). Here, we characterized the spectrum of 1324 RTK fusions with intact kinase domains in solid tumors by DNA-based high-throughput sequencing. Overall, the prevalence of RTK fusions were 4.7%, with variable frequencies and diverse genomic structures and fusion partners across cancer types. Cancer types, such as thyroid cancers, urological cancers and neuroendocrine tumors are selective in the RTK fusions they carry, while others exhibit highly complex spectra of fusion events. Notably, most RTKs were promiscuous in terms of the partner genes they recombine with. A large proportion of RTK fusions had one of the breakpoints localized to intergenic regions. Comprehensive genomic profiling revealed differences in co-mutational patterns pre- and post-TKI treatments across various RTK fusions. At baseline, multiple cases were detected with co-occurring RTK fusions or concomitant oncogenic mutations in driver genes, such as KRAS and EGFR. Following TKI resistance, we observed differences in potential on- and off-target resistance mutations among fusion variants. For example, the EML4-ALK v3 variant displayed more complex on-target resistance mechanisms, which might explain the reduced survival outcome compared with the v1 variant. Finally, we identified two lung cancer patients with MET+ and NTRK1+ tumors, respectively, who responded well to crizotinib treatment. Taken together, our findings demonstrate the diagnostic and prognostic values of screening for RTK fusions using DNA-based sequencing in solid tumors.

Experimentally Deduced Criteria for Detection of Clinically Relevant Fusion 3' Oncogenes from FFPE Bulk RNA Sequencing Data.

Drugs targeting receptor tyrosine kinase (RTK) oncogenic fusion proteins demonstrate impressive anti-cancer activities. The fusion presence in the cancer is the respective drug prescription biomarker, but their identification is challenging as both the breakpoint and the exact fusion partners are unknown. RNAseq offers the advantage of finding both fusion parts by screening sequencing reads. Paraffin (FFPE) tissue blocks are the most common way of storing cancer biomaterials in biobanks. However, finding RTK fusions in FFPE samples is challenging as RNA fragments are short and their artifact ligation may appear in sequencing libraries. Here, we annotated RNAseq reads of 764 experimental FFPE solid cancer samples, 96 leukemia samples, and 2 cell lines, and identified 36 putative clinically relevant RTK fusions with junctions corresponding to exon borders of the fusion partners. Where possible, putative fusions were validated by RT-PCR (confirmed for 10/25 fusions tested). For the confirmed 3′RTK fusions, we observed the following distinguishing features. Both moieties were in-frame, and the tyrosine kinase domain was preserved. RTK exon coverage by RNAseq reads upstream of the junction site were lower than downstream. Finally, most of the true fusions were present by more than one RNAseq read. This provides the basis for automatic annotation of 3′RTK fusions using FFPE RNAseq profiles.

Genomic Features of Solid Tumor Patients Harboring ALK/ROS1/NTRK Gene Fusions.

The fusions of receptor tyrosine kinase (RTK) involving anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1), and neurotrophic receptor tyrosine kinase (NTRK) represent the potential targets of therapeutic intervention for various types of solid tumors. Here, the genomic features of 180 Chinese solid tumor patients with ALK, ROS1, and NTRK fusions by next generation sequencing (NGS) were comprehensively characterized, and the data from 121 patients in Memorial Sloan Kettering Cancer Center (MSKCC) database were used to compare. We found that ALK, ROS1, and NTRK fusions were more common in younger female patients (p<0.001) and showed a higher expression of programmed death ligand 1 (PD-L1). The gene-intergenic fusion and the fusion with rare formation directions accounted for a certain proportion in all samples and 62 novel fusions were discovered. Alterations in TP53 and MUC16 were common in patients with RTK fusions. The mutational signatures of patients were mainly distributed in COSMIC signature 1, 2, 3, 15 and 30, while had a higher frequency in copy number variations (CNVs) of individual genes, such as IL-7R. In the MSKCC cohort, patients with fusions and CNVs showed shorter overall survival than those with only fusions. Furthermore, the differentially mutated genes between fusion-positive and -negative patients mainly concentrated on MAPK signaling and FOXO signaling pathways. These results may provide genomic information for the personalized clinical management of solid tumor patients with ALK, ROS1, and NTRK fusions in the era of precision medicine.

Abstract 848: Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance

Abstract Drug resistance remains a significant obstacle in the successful treatment of cancer, highlighting the critical need to understand how oncogenes and cancer drugs impact cell physiology and resistance development. EML4-ALK is a receptor tyrosine kinase (RTK) fusion oncogene that drives 3-7% of lung cancer. Despite potent ALK inhibitors, EML4-ALK+ cancers frequently develop resistance during therapy. Recently, it was discovered that EML4-ALK and other RTK fusions form cytoplasmic protein condensates, and that condensate formation was required for oncogenic signaling. However, whether oncogenic condensates play a role in drug responses is unclear. In this study, we applied an optogenetic technique called ‘functional profiling’ to understand how EML4-ALK condensates impact cell signal transmission and drug response. Using light-stimulated RTKs, we found that EML4-ALK condensates strongly suppress signaling through transmembrane RTKs, including through EGFR, a central receptor in resistance development. Strikingly, treatment with ALK inhibitors (ALKi) rapidly restored and hypersensitized RTK signaling. We found that EML4-ALK condensates suppress RTK signals through sequestration of the downstream adapter Grb2, which is essential for signaling through EGFR and other RTKs. The release of Grb2 from condensates resensitized RTKs within 10s of minutes of ALKi addition. Resensitized RTKs, in turn, caused sporadic RTK activation pulses throughout the cell population, and pulses originated from paracrine RTK signals released by apoptotic neighbors. We found that these paracrine signals counteracted ALK inhibitor therapy and promoted survival and drug tolerance. Blocking paracrine signals through co-treatment of ALKi with inhibitors of either EGFR or matrix metalloproteases enhanced cell killing and minimized long-term drug tolerance. Our study uncovers a role for oncogenic condensates in drug resistance signaling, reveals a novel mechanism for oncogene-induced suppression of RTK signaling, and suggests novel co-therapies to more effectively treat cancers driven by EML4-ALK and possibly other RTK fusions. Our work also demonstrates the potential of functional optogenetic profiling for drug discovery to promote cancer therapy. Citation Format: David Gonzalez-Martinez, Lee Roth, Thomas Mumford, Yael Mosse, Asmin Tulpule, Trever Bivona, Lukasz Jan Bugaj. Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 848.

Abstract 5771: The refined stratification of receptor tyrosine kinase fusions in lung cancer

Abstract Background: The overall survival of patients with the actionable receptor tyrosine kinase (TRK) fusions can be significantly prolonged after taking targeted kinase inhibitors. However, the outcomes are variable due to the different fusion types of the same driver gene. Method: Formalin fixed paraffin embedded tissues from patients with lung cancer between March 2018 and September 2021 were performed by using next generation sequencing (NGS) at Genetronhealth, a laboratory accredited by College of American Pathologists and Clinical Laboratory Improvement Amendments. Results: In a large cohort of 3768 patients with lung cancer, 348 cases harboring the actionable TKR fusions, including ALK, ROS1, RET, EGFR, MET, BRAF, NTRK1/2/3. TRK fusions can be divided into three types based on the breakpoints, partners and fusion models: single 3’ fusions, nonreciprocal fusions with the intact tyrosine kinase domain (TKD); single 5’ fusions, reciprocal fusions with the incomplete TKD; complex fusions, nonreciprocal/reciprocal fusions or nonreciprocal fusions with different partners. At the gene level, 58% (203 of 348) of TRK fusions were ALK, among which 71% of single 3’ ALK (144 of 203), 3% of single 5’ ALK (6 of 203) and 26% of complex fusions (53 of 203) were included. ROS1 fusions accounted for 14% (50 of 348) of all TRK fusions, and the proportions of single 3’ fusions, single 5’ fusions and complex fusions were 60% (30 of 50), 6% (3 of 50) and 34% (17 of 50), respectively. RET captured 19% (66 of 348) TRK fusions with 70% of single 3’ RET (46 of 66), 5% of single 5’ RET (3 of 66) and 26% of the complex fusions (17 of 66). EGFR owned 3% (9 of 348) with 33% of single 3’ EGFR (3 of 9), 33% of single 5’ EGFR (3 of 9) and 33% of the complex (3 of 9). MET, BRAF and NTRK1/2/3 shared the rest 5% of total fusions without complex type. Conclusion: Our research shows different types of TRK fusions in Chinese lung cancer patients, which can help guide the precise treatment in clinical. Citation Format: Chaoyang Liang, Lin Li, Xiaofang Liu, Qiuhua Liu, Baoming Wang, Chunyang Wang, Tonghui Ma, Fanlu Meng. The refined stratification of receptor tyrosine kinase fusions in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5771.