Kinase Domain Duplication Research Articles

Spindle Cell Lesions with Oncogenic EGFR Kinase Domain Aberrations: Expanding the Spectrum of Protein Kinase–Related Mesenchymal Tumors

EGFR aberrations are reported in a subset of myofibroblastic lesions with kinase domain duplication (EGFR-KDD) and exon 20 mutations being assigned to infantile fibrosarcomas (IFS), mesoblastic nephroma, and fibrous hamartoma of infancy (FHI), respectively. In this retrospective study, we correlated molecular findings with the histomorphology of 14 myofibroblastic lesions harboring such genetic changes identified by NGS. We additionally performed DNA methylation profiling (DNAmp) and immunohistochemistry. Lesions were from 10 males and 4 females with a mean age of 3 years (range, 0.3-14) and occurred subcutaneously in the upper limbs (n = 5), lower limbs (n = 3), back/thorax (n = 5), and the nasal cavity (n = 1). Eleven were cured by surgery, including 1 relapsed case. Two patients were lost to follow-up. One case was very recent, and the patient was biopsied. Histologically, the lesions showed a wide spectrum varying from classic FHI (n = 9) to IFS (n = 1) or lipofibromatosis-like tumors (LFT-like) (n = 2) or dermatofibrosarcoma protuberans-like (DFSP-like) (n = 1) to a predominantly myxoid spindle cell lesion (n = 1). Immunohistochemically, all neoplasms stained with CD34, whereas S100 was positive in 2/14. EGFR expression was observed in 9/10 cases. Molecularly, the IFS and 1 LFT-like harbored EGFR-KDD, whereas an exon 20 mutation was identified in all FHI, 1 LFT-like, the DFSP-like, and in predominant myxoid spindle cell lesion. By DNAmp, all but 2 cases formed a well-defined cluster, demonstrating that these lesions are also epigenetically related. In conclusion, EGFR kinase domain aberrations found in FHI, IFS, LFT-like, DFSP-like, and a spindle cell lesion with a predominant myxoid stroma of children and adolescents showed that these neoplasms with a broad morphologic spectrum belong to the group of protein kinase–related lesions with a distinct epigenetic signature. Molecular analyses, including DNAmp, help to identify and characterize this emerging category and become mandatory when targeted treatment is considered.

Colorectal cancer harboring EGFR kinase domain duplication response to EGFR tyrosine kinase inhibitors.

Epidermal growth factor receptor kinase domain duplication (EGFR-KDD) is a rare, recurrent oncogenic variant that constitutively activates EGFR in non-small-cell lung cancer. Herein, we report the case of a 70-year-old man with resectable colorectal adenocarcinoma who underwent surgery followed by adjuvant therapy. He relapsed with multiple liver metastases and received standard chemotherapy until his disease became refractory. Comprehensive genomic profiling of his postoperative colorectal cancer tissue revealed EGFR-KDD. He was treated with an EGFR tyrosine kinase inhibitor (TKI), afatinib and achieved a partial response (- 55%) after 8 weeks; however, he developed massive malignant ascites after 13 weeks. Osimertinib, another EGFR-TKI, controlled his tumors for 9 months. Patient-derived cancer organoids from his malignant ascites confirmed sensitivity to EGFR-TKIs. The findings suggest that EGFR-TKIs can be a potential treatment option for this molecular subgroup.

439 Extracellular-in-frame deletions and kinase domain duplications are novel, gain-of-function mutations in fibroblast growth factor receptor genes in cancer

OBJECTIVES/GOALS: There are gain-of-function genomic alterations in FGFR genes that guide personalized treatment in some patients with cholangiocarcinoma (10%) and bladder cancer (30%) who can benefit from targeted therapies. We sought to evaluate other genomic alterations in cancer involving FGFRs and assess whether they are gain-of-function. METHODS/STUDY POPULATION: We collaborated with Foundation Medicine Inc (FMI), for the assessment of 300,000 sequenced tumors and a retrospective analysis of recent publications, to identify novel candidate FGFR alterations. We propose to transiently transfect HEK293T cells with an empty vector (EV), FGFR1-4 wild-type (WT), and these variants and use a luminescent-proximity based high-throughput assay, AlphaLISA, and Western blot to assess FGFR and phosphorylated downstream signaling proteins, FRS2, AKT and ERK, and their sensitivity to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200. RESULTS/ANTICIPATED RESULTS: Through our collaboration we identified >100 novel candidate FGFR1-4 variants of unknown significance (VUS) including extracellular-in-frame deletions (EIDs), kinase domain duplications (KDDs), insertions/deletions (INDELs), short number variants (SNVs), and truncations. Immunoblot analysis confirmed the presence of desired EV, FGFR WT, and VUS’ in HEK293T cells. We anticipate the FGFR EIDs and KDDs to display an increased presence of in the respective pFGFR, pFRS2, pERK, and pAKT as compared to the EV and FGFR WT by both immunoblot and AlpahLISA analysis. Additionally, we anticipate the VUS’ to be sensitive to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200 using the AlphaLISA assay. DISCUSSION/SIGNIFICANCE: These findings suggest that the novel FGFR VUS’ are capable of constitutive activation of FGFR kinase activity, and they preliminary demonstrate that these newly identified FGFR alterations are therapeutically targetable. Thus, providing rationale for further clinical evaluation to identify new cohorts of FGFR inhibitor responders.

Combination Therapy with EGFR Tyrosine Kinase Inhibitors and TEAD Inhibitor Increases Tumor Suppression Effects in EGFR Mutation-positive Lung Cancer.

EGFR-tyrosine kinase inhibitors (TKI) are the first-line therapies for EGFR mutation-positive lung cancer. EGFR-TKIs have favorable therapeutic effects. However, a large proportion of patients with EGFR mutation-positive lung cancer subsequently relapse. Some cancer cells survive the initial treatment with EGFR-TKIs, and this initial survival may be associated with subsequent recurrence. Therefore, we aimed to overcome the initial survival against EGFR-TKIs. We hypothesized that yes-associated protein 1 (YAP1) is involved in the initial survival against EGFR-TKIs, and we confirmed the combined effect of EGFR-TKIs and a YAP1-TEAD pathway inhibitor. The KTOR27 (EGFR kinase domain duplication) lung cancer cell lines established from a patient with EGFR mutation-positive lung cancer and commercially available PC-9 and HCC827 (EGFR exon 19 deletions) lung cancer cell lines were used. These cells were used to evaluate the in vitro and in vivo effects of VT104, a TEAD inhibitor. In addition, YAP1 involvement was investigated in pathologic specimens. YAP1 was activated by short-term EGFR-TKI treatment in EGFR mutation-positive lung cancer cells. In addition, inhibiting YAP1 function using siRNA increased the sensitivity to EGFR-TKIs. Combination therapy with VT104 and EGFR-TKIs showed better tumor-suppressive effects than EGFR-TKIs alone, in vitro and in vivo. Moreover, the combined effect of VT104 and EGFR-TKIs was observed regardless of the localization status of YAP1 before EGFR-TKI exposure. These results suggest that combination therapy with the TEAD inhibitor and EGFR-TKIs may improve the prognosis of patients with EGFR mutation-positive lung cancer.

Liquid-biopsy detection of FGFR2 and other actionable rearrangements in GI malignancies.

4085 Background: Actionable rearrangements (RE) represent an emerging therapeutic target for GI malignancies. However, there remains uncertainty whether liquid biopsy (LBx) assays testing circulating tumor DNA (ctDNA) can detect RE with the same fidelity as tissue biopsy. Here we report the performance of ctDNA-based comprehensive genomic profiling (CGP) for the detection of RE leveraging an FDA-approved >300-gene platform, with a focus on data from patients with GI malignancies. Methods: An institutional research database of clinical CGP results for tissue biopsy (TBx, FoundationOneCDx) and LBx (FoundationOneLiquid CDx) from patients with cancers of the GI tract was reviewed. Sensitivity for FGFR2 RE was studied in patients with cholangiocarcinoma (CCA) or carcinoma of unknown primary (CUP) and both TBx and LBx CGP available. Results: Across 7870 GI LBx, 1094 predicted pathogenic RE were detected in 826 cases (10%) including 283 oncogenic kinase RE, 686 inactivating RE, and 125 other gain-of-function RE. FGFR2 was the most frequently rearranged gene, enriched in CCA (4.4%, 37/833) and stomach (3.0%, 11/368). EGFR RE (38) occurred across 6 cancer types and included 8 fusions, 15 c-terminal truncations, 4 intragenic deletions/inversions, and 3 kinase domain duplications. BRAF RE (31) were detected among in colorectal (22) and pancreas (9) cases. LBx detected 44 exon 3 skipping (protein stabilizing) RE in CTNNB1 (beta-catenin) across all samples. Other frequent activating RE were detected in MYC (36), FGFR3, RET, ROS1 (21 each), and ALK (13). Potentially targetable inactivating RE were detected in BRCA1 (28), ARID1A (24), STK11 (22), PTEN (17), TSC2 (14), and BRCA2 (8). Focusing on CCA, FGFR2 RE were more prevalent in cases with elevated (≥1%) ctDNA tumor fraction (TF, 26/342, 7.6%), matching the prevalence in TBx (7.8%, 505/6,492) with a similar spectrum of fusion partners seen for both TBx and LBx (30% BICC1, 70% rare for both). In 13 CCA/CUP patients positive for FGFR2 RE by TBx, LBx detected 12 (92%) including 8 different fusion partners; the false negative case had TF <1%. In 11 CCA LBx samples with FGFR2 resistance mutations detected, LBx CGP successfully detected a driver FGFR2 RE in 10 and driver C382R mutation in 1. Conclusions: Rearrangements are represented across GI malignancies, including many that result in known oncogenic drivers that may be targetable with available therapies. We observe reliable detection of FGFR2 fusions in ctDNA, in contrast to some prior publications. ctDNA represents a pragmatic analyte for detection of rearrangements and other actionable alterations, offering timely result return when tissue is inadequate or unavailable. [Table: see text]

Abstract 3918: Kinase domain duplications of FGFR1 and MET are potential therapeutic targets

Abstract Background: Kinase domain duplication (KDD) is recently recognized as an oncogenic driver. Therapeutic potential has been established in EGFR-KDDs in non-small cell lung cancer, and our group recently showed resistance mechanism in lung cancers harboring EGFR-KDDs treated with EGFR tyrosine kinase inhibitors (TKIs). However, the frequency and oncogenic role of KDDs in other receptor tyrosine kinases has been elusive. Methods: To investigate the frequency of KDDs in advanced cancer patients, we developed in-house algorithm to detect KDDs in high-depth panel sequencing dataset and evaluated its performance using large-scale panel sequencing data from Seoul National University Hospital (SNUH). In short, the algorithm collects reads with soft clips from a modified GATK best practice pipeline for clinical NGS data processing. The collected reads are then grouped according to their matched position and insert size, and evidences of each group (number of splitting and spanning reads) are collected to yield a set of initial candidates. Finally, the initial candidates were manually inspected after quality control by removing false-positive candidates, such as non-target genes or consistent findings. Then, we established cell lines transfected with KDDs of FGFR1 and MET to validate oncogenic role of identified KDDs in the cohort. Using these transformed cell lines, we investigated carcinogenicity with cell viability assay, immunoblot assay and colony formation assay. FGFR TKIs and MET TKIs were used to uncover the possibility of therapeutic targets in the KDDs. Results: Using the in-house algorithm, we identified FGFR1-KDDs from brain tumors (n=4), EGFR-KDDs from lung (n=1) and brain (n=2) tumors, and MET-KDD from a lung tumor (n=1) in a total of 3,932 cancer samples, with 0.2% frequency. All three kinds of KDDs contained full-length of kinase domains of each gene. Ba/F3 cells transfected with the FGFR1-KDD constructs proliferated well without IL-3 (1239- and 1019-fold higer than wild-type FGFR1), while wild-type FGFR1 Ba/F3 cells did not. This growth potential was diminished by FGFR TKIs BGJ398 (IC50, 0.063 ± 0.065nM), PD173074 (IC50, 1.498 ± 1.021nM), and ponatinib (IC50, 5.69 ± 3.05nM). Reduced expressions of downstream pathway proteins including phospho-FGFR1, phospho-Akt, and phospho-Erk were confirmed by immunoblot assays. NIH-3T3 cells transfected with MET-KDD construct also showed growth advantage compared to wild-type MET NIH-3T3 cells. Capmatinib, a MET TKI, inhibited colony formation of MET-KDD NIH-3T3 cells with reduced phospho-MET expression. Conclusion: In this study, we validated newly developed KDD-detection computational methods using targeted DNA sequencing data. Using this algorithm, we found 0.2% of KDDs in 3,932 cancer samples, including EGFR, FGFR1, and MET. In addition to EGFR-KDDs, our study reveals that both FGFR1- and MET-KDD can be oncogenic and therapeutic targets in brain and lung cancer patients. Citation Format: Chaelin Lee, Sungyoung Lee, Hongseok Yun, Jeonghwan Youk, Tae Min Kim, Soyeon Kim, Bhumsuk Keam, Miso Kim, Dong-Wan Kim. Kinase domain duplications of FGFR1 and MET are potential therapeutic targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3918.

Clinical and molecular characteristics of kinase domain duplications across diverse cancer types in the Chinese population.

Kinase domain duplications (KDDs) have recently been recognized as oncogenic mutations and possible association with drug resistance in cancers. Here, targeted sequencing was performed with the tumor tissue and/or plasma from 65 cancer patients with KDDs. Intact KDDs were identified in approximately 0.1% of the total population across multiple cancer types. EGFR KDD was first identified in colorectal cancer and breast cancer, whereas FGFR2 KDD was first identified in gastric cancer. Tumors with EGFR KDD displayed lower concurrent TP53 gene alterations (p= 0.03) and slightly higher chromosome instability (p= 0.27) compared to tumors with non-EGFR-KDDs. Immune pathway analysis further revealed the enrichment of the cytokine receptors pathway (93%) in the KDD carriers. Hyperprogression-related gene mutations were identified in four cases. Collectively, our data revealed the genomic features of KDD alterations in a multi-cancer cohort, providing more information for the potential treatment application in the KDD carriers.

Abstract 2674: Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and > 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values < 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [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 2674.

Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis.

A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

Infantile fibrosarcoma with an EGFR kinase domain duplication: Underlining a close relationship with congenital mesoblastic nephroma and highlighting a similar morphological spectrum

Infantile fibrosarcoma (IFS) and congenital mesoblastic nephroma (CMN) are locally aggressive tumors primarily occurring in infants. Both IFS and the cellular subtype of CMN show overlapping morphological features and an ETV6-NTRK3 fusion, suggesting a close relationship. An activating alteration of EGFR, based on an EGFR kinase domain duplication (KDD), occurs in a subset of CMNs lacking an NTRK3 rearrangement, especially in the classic and mixed type. So far no EGFR-KDDs have been detected in IFS.We describe four pediatric tumors at the extremities (leg, n = 2; foot and arm n = 1) with histological features of IFS/CMN. Two cases showed classic IFS morphology while two were similar to classic/mixed type CMN. In all cases, an EGFR-KDD was identified without detection of a fusion gene. There were no abnormalities of the kidneys in any of the patients.This is the first description of IFS with an EGFR-KDD as driver mutation, supporting that IFS and CMN are similar lesions with the same morphological and genetic spectrum. Pathologists should be aware of the more fibrous variant of IFS, similar to classic/mixed type CMN. Molecular analyses are crucial to treat these lesions adequately, especially with regard to the administration of tyrosine kinase inhibitors.

113P The analysis of EGFR fusions characteristics in Chinese solid tumor patients

Oncogenic mutations in the epidermal growth factor receptor (EGFR) serve as important predictive biomarkers in non-small cell lung cancer (NSCLC). The constitutive activity and sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in lung cancer with small in-frame deletions in exon 19, mutations in exon 21 in the EGFR tyrosine kinase domain had been conferred. Atypical EGFR aberrations, including EGFR kinase domain duplications (KDD) and EGFR fusions (EGFR-RAD51, EGFR-SEPT14, KIF5B-EGFR) had been identified.

Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications

Abstract Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode. Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.

Acquired Resistance Mechanism of EGFR Kinase Domain Duplication to EGFR TKIs in Non-Small Cell Lung Cancer.

PurposeEpidermal growth factor receptor kinase domain duplication (EGFR-KDD) is a rare and poorly understood oncogenic mutation in non–small cell lung cancer (NSCLC). We aimed to investigate the acquired resistance mechanism of EGFR-KDD against EGFR-TKIs.Materials and MethodsWe identified EGFR-KDD in tumor tissue obtained from a patient with stage IV lung adenocarcinoma and established the patient-derived cell line SNU-4784. We also established several EGFR-KDD Ba/F3 cell lines: EGFR-KDD wild type (EGFR-KDDWT), EGFR-KDD domain 1 T790M (EGFR-KDDD1T), EGFR-KDD domain 2 T790M (EGFR-KDDD2T), and EGFR-KDD both domain T790M (EGFR-KDDBDT). We treated the cells with EGFR tyrosine kinase inhibitors (TKIs) and performed cell viability assays, immunoblot assays, and ENU (N-ethyl-N-nitrosourea) mutagenesis screening.ResultsIn cell viability assays, SNU-4784 cells and EGFR-KDDWT Ba/F3 cells were sensitive to 2nd generation and 3rd generation EGFR TKIs. In contrast, the T790M-positive EGFR-KDD Ba/F3 cell lines (EGFR-KDDT790M) were only sensitive to 3rd generation EGFR TKIs. In ENU mutagenesis screening, we identified the C797S mutation in kinase domain 2 of EGFR-KDDBDT Ba/F3 cells. Based on this finding, we established an EGFR-KDD domain 1 T790M/domain 2 cis-T790M+C797S (EGFR-KDDT/T+C) Ba/F3 model, which was resistant to EGFR TKIs and anti-EGFR monoclonal antibody combined with EGFR TKIs.ConclusionOur study reveals that the T790M mutation in EGFR-KDD confers resistance to 1st and 2nd generation EGFR TKIs, but is sensitive to 3rd generation EGFR TKIs. In addition, we identified that the C797S mutation in kinase domain 2 of EGFR-KDDT790M mediates a resistance mechanism against 3rd generation EGFR TKIs.

Structure\u2013function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting

Mechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication of EGFR exons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence of ERBB family KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establish ERBB KDDs as recurrent oncogenic events in multiple cancers.

P89.09 The Landscape of Kinase Domain Duplication (KDD) in Chinese Lung Cancer Patients

P89.09 The Landscape of Kinase Domain Duplication (KDD) in Chinese Lung Cancer Patients

The landscape of kinase domain duplication in Chinese lung cancer patients.

BackgroundKinase domain duplication (KDD) is a special type of large genomic rearrangement (LGR), occurring in the kinase domain of protein kinase genes. KDD of some lung cancer driver genes, such asEGFRKDD, has been identified and implicated to be oncogenic in non-small cell lung cancer (NSCLC). The present study aims to interrogate the spectrum of KDD occurring on classic driver genes in Chinese lung cancer patients without the presence of classic lung cancer driver mutations.MethodsWe retrospectively enrolled 10,525 Chinese lung cancer patients who met the following inclusion criteria; (I) do not carry classic lung cancer driver mutations in any of the 8 driver genes and (II) tyrosine kinase inhibitor (TKI)-naïve. Capture-based targeted sequencing was performed on tissue or plasma samples. LGR and KDD were identified by using in-house analysis scripts. The prevalence and distribution of LGR and KDD in our cohort were analyzed.ResultsThe median age of the cohort was 64 years with 68.7% being male. Among all patients, 23.2% and 51.8% were diagnosed with stage III and IV disease respectively. We identified 43 cases (0.41%) harboring LGR in one of the driver genes (EGFR/ERBB2/ALK/RET/ROS1/MET/BRAF), with 24 (0.23%) patients harboring KDD. Of the patients harboring KDD, a majority (n=19) harbored canonical EGFR-KDD involving exons 18–25, whilst one patient harbored duplications of EGFR exons 18–26. There were three MET-KDD patients; in two, the alteration occurred in exons 15–21 and in one, the alteration occurred in exons 3–21. One patient harbored RET-KDD involving exons 12–18. KDD showed a comparable prevalence in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (0.33% vs. 0.11%, P=0.118). Nineteen non-KDD LGRs, spanning six genes including EGFR (n=6), MET (n=3), ALK (n=4), ROS1 (n=2), ERBB2 (n=2) and BRAF (n=2), were found, each occurring in one patient. The prevalence of LGR in LUADs and LUSCs was comparable (0.55% vs. 0.38%, P=0.452).ConclusionsWe observed a prevalence of 0.41% and 0.23% for LGR and KDD, respectively. Twenty-four different LGR alterations, including 5 KDDs and 19 non-KDD LGRs, were observed. KDDs mainly occurred in EGFR involving exons 18–25 and non-KDD LGRs were distributed more randomly. The prevalence of LGR/KDD in LUSCs and LUADs was comparable.

Identification of kinase domain duplications across diverse cancer types in Chinese population by comprehensive genomic profiling (CGP).

e13522 Background: Kinase domain duplication (KDD) has recently been recognized as oncogenic and targetable mutations in some cancers. EGFR KDD was identified as an oncogenic driver in lung cancer showing partial response to EGFR TKI. BRAF KDD was reported in diverse tumor types with clinical response to RAF-targeted therapy. We retrospectively investigated the prevalence of KDDs in multiple cancer types of a large Chinese population. Methods: 50742 unique cancer cases were analyzed by comprehensive genomic profiling (CGP). DNA was extracted from formalin-fixed paraffin-embedded specimens (FFPEs), fresh tissue, blood or plasma samples and sequenced with gene panels targeting 400+ cancer-relevant genes. Among them, 53 cases were detected with KDD of various kinases. Results: In this Chinese cohort, KDD was identified in 0.1% of the total population across multiple cancer types including lung cancer (39), breast cancer (5), gastric cancer (3), colorectal cancer (2), mucoepidermoid carcinoma (1), unknown (3). The median age at diagnosis was 54 which was younger than the 60 yrs median age of total population. The distribution of KDDs was in EGFR(34) MET(5), JAK1(2), BRAF(2), FGFR2(2), FGFR1(1), JAK2(1), LYN(1), MAP3K1(1), RAF1(1), RET(1), AKT3(1), and CDK8(1). Thirty-one lung cancer cases were detected with EGFR-KDD, including kinase duplications of exon18-25 (22), exon17-25 (6), exon18-26 (2), exon14-26(1). Three patients with EGFR-KDD exon18-25 showed partial anti-tumor response to target therapy. MET-KDD was exclusively found in lung cancer involving the duplication of MET exon15-21 (2), exon14-17 (1), exon15-16 (1) and exon12-21 (1) while FGFR1/2-KDD was observed only in gastric cancer. Two female patients with breast cancer were detected with JAK1-KDD at age of 45 and 37, respectively. The canonical BRAF-KDD of exon 10-18 was identified in one female patient diagnosed of lung adenocarcinoma at age of 49. Frequently altered genes in patients with KDD were TP53(72%), EGFR (23%), FAT1(13%), BRCA1(10%). MCL1 amplification, a known oncogenic alteration, was identified in fifteen patients (11 EGFR-KDD,2 MET-KDD, 1 BRAF-KDD, 1 JAK2-KDD), representing the most common copy number variation observed. Conclusions: Kinase KDDs were rare but potentially oncogenic mutations in diverse cancer types with clinical outcome of EGFR-KDD to target therapy in lung cancer. Cancer-type specific KDDs were identified including MET-KDD in lung cancer, FGFR1/2-KDD in gastric cancer and JAK1-KDD in breast cancer.

Recurrent EGFR alterations in NTRK3 fusion negative congenital mesoblastic nephroma

ObjectivesTo identify oncogenic driver mutations in congenital mesoblastic nephroma (CMN) cases lacking ETV6-NTRK3 fusion and discuss their diagnostic value. DesignThe institutional pathology database was queried for cases with a morphologic diagnosis of CMN. Cases positive for ETV6 rearrangement or with unavailable blocks were excluded. Four cases met the inclusion criteria and were sequenced by next-generation sequencing. Three additional cases were contributed by our collaborators. ResultsThree of four internal cases harbor an EGFR kinase domain duplication (KDD), which is known to be oncogenic yet exceedingly rare in other histologies. All three outside cases are positive for EGFR alterations, including KDD in two and a splicing site mutation in one. The splicing site mutation is predicted to be EGFR activating. One of the outside cases was a retroperitoneal mass without a clear site of origin. A diagnosis of CMN is suggested based on exclusion of differential diagnoses by expert consultation and detection of EGFR KDD. ConclusionsEGFR activation, predominantly via EGFR KDD, is a common recurrent genetic alteration in CMN lacking NTRK3 fusions. CMN can be molecularly classified into NTRK3 fusion type, EGFR activation type and others.

Abstract GMM-025: ERBB FAMILY GENOMIC ALTERATIONS IN GYNECOLOGICAL MALIGNANCIES

Abstract Activation of HER2 or EGFR signaling is a common, targetable oncogenic mechanism in solid tumors such as breast, gastroesophageal, lung and colorectal carcinomas. In addition, ERBB3 activation has been shown to confer sensitivity to HER2-targeted therapies, and ERBB4 mutation is reported a mechanism of resistance. From a database of 14,692 comprehensive genomic profiles for both common and rare gynecological malignancies, we extracted 9,121 ovarian tumor profiles and evaluated the prevalence of genomic alterations (GA) in the ERBB family (EGFR, ERBB2, ERBB3, ERBB4), including base substitutions, small indels, amplification (AMP), and rearrangements (RE). At least 50ng of DNA was extracted from FFPE specimens and sequenced to high, uniform median coverage (>500X) by hybridization capture of exons from up to 406 cancer-related genes and select introns from up to 31 genes commonly rearranged in cancer. For some samples, RNA sequencing of 265 genes was also performed. Tumor mutational burden (TMB, mutations/Mb [mut/Mb]) was determined on ~1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) and overall genomic loss of heterozygosity was evaluated for subsets of the samples. Of 9,121 tumors, most were serous carcinomas (n = 6,847). A total of 615 (6.7%) tumors had ≥1 oncogenic GA in an ERBB gene, with 40 (0.4%) having ≥2 ERBB GA. ERBB+ were most often mucinous (15.9%) or clear cell (13.9%) carcinomas. Serous carcinomas were ERBB+ in 4.3-7.1% of samples. Patient age was comparable in both the ERBB+ and ERBB- tumors (median 61 y vs. 61 y, range 19-94 y vs. 2-95 y). ERBB2 (HER2) AMP was the most common ERBB GA; 2.7% of serous carcinomas had amplified ERBB2. GA distribution by ERBB gene was as follows: ERBB2 4.5% (n=394; of those AMP were 78.4%, n=309), ERBB3 0.8% (n=87; AMP 50.6%, n=44), EGFR 0.9% (n=90; AMP 45.6%, n=41), and ERBB4 0.6% (n=66; AMP 65.1%, n=43). Known or potential oncogenic rearrangements, fusions, and splice site GA were observed in both ERBB2 (n=20) and EGFR (n=10), including a kinase domain duplication in EGFR and exon 16 deletions in ERBB2. Although ERBB2 AMP is concordant with HER2+ by IHC or FISH, 70/615 (11.4%) HER2-driven samples harbored targetable, oncogenic GA that would not be detected by these methods, including the well-characterized A775_G776insYVMA (n=13), S310Y/F (n=10), and V842I (n=8). In ERBB+ tumors, the most commonly co-occurring GA affected TP53 (76.6%), PIK3CA (19.5%), MYC (15.7%), TERC (14.6%), ARID1A (18.3%), and CCNE1 (20.5%). Pathways likely to confer some level of resistance to ERBB-targeted therapies were often mutated: PI3K-AKT-MTOR 35.0% (n=215), RAS-RAF-MEK 18.2% (n=112), and FGFR 5.5% (n=34). ERBB+ tumors had slightly higher TMB scores: median 3.5 mut/Mb vs 2.6 mut/Mb, and average 6.7 vs 3.7 mut/Mb. Of 502 samples analyzed for microsatellite instability, 12 were MSI high. In conclusion, nearly 7% of ovarian tumors harbor potentially targetable GA in an ERBB family gene, including HER2 and EGFR. Of these, 49.6% of samples would not be positive by HER2 IHC or FISH. Further exploration into the efficacy of HER2- or EGFR-targeted therapies, and the effects of co-altered pathways, in ovarian carcinomas may be promising. Citation Format: Laurie M Gay, Jo-Anne Vergilio, J Keith Killian, Shakti Ramkissoon, Eric Severson, Sugganth Daniel, Amanda Hemmerich, Jeffrey S Ross, Julia A Elvin. ERBB FAMILY GENOMIC ALTERATIONS IN GYNECOLOGICAL MALIGNANCIES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-025.

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency