Mutation Fusion Research Articles

Partial Response to Treatment with ALK Inhibitor in a Patient With SQSTM1-ALK Fusion Positive Lung Adenocarcinoma.

Lung cancer is the second most common cancer worldwide and the leading cause of cancer deaths; non-small cell lung cancer (NSCLC) constitutes about 85% of lung cancer cases, with ALK fusions representing 3-6% of them. The SQSTM1-ALK fusion is a rare finding in NSCLC, accounting for only 1.1% of ALK rearrangements. We present a case of lung adenocarcinoma with documentation of SQSTM1-ALK fusion that showed a partial response to alectinib. This case details the clinical course of a 71-year-old, non-smoking woman with no significant medical history who presented with confusion, aphasia and multiple cerebral lesions detected on imaging. Further investigations revealed a stage IV lung adenocarcinoma with metastases to the brain and adrenal gland. Molecular profiling identified a rare SQSTM1-ALK fusion mutation alongside other genetic abnormalities, including low programmed death-ligand 1 expression and ROS1 kinase protein presence. Treatment with alectinib, initiated based on the identified ALK fusion, resulted in significant tumour regression in the lungs and complete resolution of the adrenal mass, as evidenced by follow-up imaging and clinical assessments. This case highlights the efficacy of alectinib in treating rare ALK fusion variants in and underscores the importance of comprehensive molecular profiling in guiding targeted therapy decisions. Recognition of rare ALK fusions This case highlights the importance of identifying rare ALK fusions, such as SQSTM1-ALK, in non-small cell lung cancer (NSCLC), which can guide personalised treatment strategies.Utility of advanced molecular diagnostics The use of next-generation sequencing alongside immunohistochemistry is crucial for accurately detecting ALK and ROS1 rearrangements, avoiding false positives and enabling the identification of druggable mutations.Impact of personalised medicine This case reinforces the value of personalised medicine in NSCLC, where molecular profiling can uncover unique genetic alterations, allowing for more tailored and potentially more effective treatments.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Descriptive Analysis of Common Fusion Mutations in Papillary Thyroid Carcinoma in Hungary

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto’s disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto’s disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

8788 Implications of NTRK gene fusion in Papillary thyroid cancer

Abstract Disclosure: W. Medina-Torres: None. L. El Musa Penna: None. J. Segarra-Villafane: None. L.R. Sepulveda-Garcia: None. Z. Maisonet -Feliciano: None. I.C. Arroyo: None. M. Ramirez: None. M. Alvarado: None. L.A. Gonzalez-Rodriguez: None. V.J. Carlo: None. Thyroid cancer (TC) is the most common endocrine cancer. Papillary thyroid carcinoma (PTC) is the predominant contributor of this occurrence, comprising approximately 80% of all thyroid carcinomas. PTC has a good prognosis with a 5-year survival rate of more than 90% after resection. Nonetheless, factors such as tumor size, multifocality, nodal and distant metastases, characterize more aggressive cases including tall cell variant, columnar cell variant, and hobnail variant Aside from staging parameters, PTC behavior is mostly determined by morphologic variant. This is the case of 31 y/o female without known past medical history who was evaluated at endocrinology clinic due to a fine needle thyroid aspiration (FNA) positive for PTC, tall cell variant. Total thyroidectomy with central compartment dissection was performed. Pathology report presented a main tumoral mass of 3 cm but permeating multinodular pattern involving the entire gland with focal extrathyroidal extension, positive margins and six level VI positive lymph nodes. As per report, the tumor presented both papillary and follicular patterns of growth and other findings including oncocytic change, foci of clear cell change, low mitotic rate, and most notably "reverse polarization”. This pattern has the pathological features suggestive of the novel NTRK gene fusion mutation. The patient was treated with radioactive iodine (RAI) and one year post treatment there was evidence of elevated Thyroglobulin level at 904 ng/mLand thyroglobulin Ab at 2.48 IU/mL. A WBS +SPECT was done resulting in small uptake at left posterior thyroid bed and pulmonary intake. A chest CT revealed multiple bilateral sub centimeter nodules. Based on this result, a second course of RAI was given. Follow up neck ultrasound and chest CT presented persistent left level VI lymph nodes, increased amount of bilateral sub-centimeter pulmonary nodules and new thoracic(T11) lesion. FNA of left neck lymph nodes were positive for metastatic PTC for which left neck dissection was performed. Evaluation for actionable somatic mutations was requested based on resistance behavior, remarkable for TRIM33-NTRK1 chromosomal rearrangement. Patient was evaluated by Oncology service who started Larotrectinib treatment since it was approved for NTRK driven malignancies. Six-month post therapy patient achieved clinical and biochemical improvement. In the thyroid, NTRK-driven malignancies are rare and aggressive but treatable, in the evolving genomic era. As reviewed in the literature, NTRK fusion-positive TC exhibits distinctive clinicopathological features that could aid in identifying potential patients. Thus, in such patients who have PTC with non-classic pathologic patterns, specific molecular profile of the main tumor may have significant prognostic value that must be integrated into stratification system in order to provide target therapy on time. Presentation: 6/3/2024

A case of neoadjuvant targeted therapy with pralsetinib for locally advanced lung adenocarcinoma with RET fusion mutation

This case report details the successful treatment of a 68-year-old male patient with locally advanced RET-rearranged lung adenocarcinoma using neoadjuvant pralsetinib. The patient initially presented with a suspicious right upper lobe nodule, which was later diagnosed as lung adenocarcinoma following genetic testing that revealed a RET exon 12 fusion. After 2 months of neoadjuvant treatment with pralsetinib, a significant radiological response was observed, with a reduction in tumor size and metabolic activity. Subsequently, the patient underwent video-assisted thoracoscopic right upper lobectomy and mediastinal lymph node dissection. Postoperative pathological analysis revealed a major pathological response, with only 5% residual tumor cells in the primary lesion and no viable tumor cells in the lymph nodes. Postoperative pathological staging of TNM was ypT1aN0M0, stage IA1(AJCC, 8th edition). The patient recovered well after surgery, demonstrating the potential efficacy of neoadjuvant pralsetinib in locally advanced RET-rearranged lung adenocarcinoma. However, further clinical validation is required to establish the role of neoadjuvant targeted therapy and postoperative adjuvant therapy in this patient population.

NTRK Fusion as an Acquired Mechanism of Resistance to Selpercatinib in RET Positive Thyroid Cancer

Background: Rearrangement during Transfection (RET) proto-oncogene is involved in the pathogenesis of various thyroid cancer subtypes and drugs that block the RET tyrosine kinase pathways are used in the management of locally advanced and metastatic Medullary Thyroid Cancer (MTC). Acquired resistance to RET inhibitors likely occurs via two different mechanisms: (1) On-target mutations that prevent drug binding and (2) Activated alternative mechanisms which bypass the targeted kinase. We present a unique mechanism of selpercatinib resistance via secondary NTRK fusion mutation in a case of RET altered medullary thyroid cancer. Case Presentation: The patient is a 72-year-old female with stage III metastatic MTC who developed recurrence with newly found RET mutation 7 years after complete surgical resection. Despite initial stable response with selpercatinib for 2 years she rapidly progressed with widespread disease. Cell-free DNA (cfDNA) testing done at the time of progression revealed an NTRK1 fusion. Unfortunately, the patient succumbed to the disease despite starting targeted therapy with larotrectinib. Conclusion: This case highlights the importance of further investigation into mechanisms of resistance to RET inhibitors and drug studies directed towards overcoming them

Establishment of genomic RNA reference materials for BCR-ABL1 P210 measurement.

Quantitation of BCR-ABL1 with thequantitative reverse transcriptase polymerase chain reaction (RT-PCR) is very important in monitoring chronic myeloid leukemia (CML), which relies on an RNA reference material. A genomic RNA reference material (RM) containing the BCR-ABL1 P210 fusion mutation was developed, and an absolute quantitative method based on one-step reverse transcription digital PCR (RT-dPCR) was established for characterizing the RM. The proposed dPCR method demonstrates high accuracy and excellent analytical sensitivity, as shown by the linear relationship (0.94 < slope < 1.04, R2≧0.99) between the measured and nominal values of b2a2, b3a2, and ABL1-ref within the dynamic range (104-101 copies/reaction). Homogeneity and stability assessment based on dPCR indicated that the RM was homogeneous and stable for 24months at -80°C. The RM was used to evaluate inter-laboratory reproducibility in eight different laboratories, demonstrating that participating laboratories could consistently produce copy concentrations of b3a2 and ABL1-ref, as well as the BCR-ABL1/ABL1 ratio (CV < 2.0%). This work suggests that the RM can be employed in establishing metrological traceability for detecting mutations in the BCR-ABL1 fusion gene, as well as in quality control for testing laboratories.

1277P Optimising targeted therapy in NSCLC: A comprehensive analysis of oncogenic fusion mutations and co-mutation landscapes

1277P Optimising targeted therapy in NSCLC: A comprehensive analysis of oncogenic fusion mutations and co-mutation landscapes

MAX germline mutation-associated pheochromocytoma-paraganglioma syndrome: multiple endocrine neoplasia type 5

The individualized precision management of hereditary pheochromocytoma (PHEO) and paraganglioma (PGL) syndromes (PPGLs) based on molecular diagnosis and molecular subtype is becoming more popular. The newly discovered MAX germline mutation-associated PPGLs are autosomally dominant and rare. To raise awareness and explore the effective management of individual diagnosis and treatment, the relevant literature published between January 2011 and February was systematically reviewed. There were a total of 101 patients in the 77 families, involving all 5 exons, containing 44 types of MAX germline mutations and mostly concentrated in exons 3 and 4 (64.4%), the main mutations were nonsense mutations and missense mutations (72.3%), and some were large fragment deletions or insertions, intron variant, gene fusion mutations were relatively infrequent. Furthermore, about 10% of the patients had a paternal parent-of-origin effect. Among the 101 patients, 96 (95.0%) developed PHEO including 15 metastatic PHEO, 61 bilateral PHEO and 35 unilateral PHEO. The age of diagnosis was (31.7±10.9) years (range: 13 to 80 years). The male to female ratio was 1.2∶1. Eleven were accompanied with chest and abdominal PGL. Eight (7.9%) were accompanied by functional pituitary adenoma. And 12 (11.9%) developed other neuroendocrine tumors (NET), of which 8 were accompanied by PHEO, including 4 hyperparathyroidism, 1 gangliocytoma and neuroblastoma, 1 pancreatic NET, 1 medullary thyroid carcinoma and 1 C cell hyperplasia. Six presented concomitant non-NET, including 1 tongue squamous cell carcinoma, 1 papillary thyroid carcinoma, 1 prostate cancer, 1 renal oncocytoma, 1 breast cancer with renal oncocytoma, and 1 thoracic chondrosarcoma with multifocal adenocarcinoma of lung. The remaining 5 cases (5.0%), including 4 other NET (2 ganglioblastoma, 1 abdominal neuroblastoma and 1 pancreatic NET) and 1 asymptomatic child, did not present PHEO. The MAX germline mutation may cause a novel multiple endocrine neoplasia, which can be described as type 5. A comprehensive baseline assessment of neural crest cell-derived diseases such as PPGL, pituitary adenoma, hyperparathyroidism, and/or gangliocytoma (neuroblastoma) was recommended for all people with MAX germline mutations, and the risk of bilateral and/or metastatic PHEO should also be considered. In contrast, patients with PPGLs combined with other NET, such as functional pituitary adenoma, should undergo genetic testing and pedigree screening that includes at least the MAX gene.

Bone-metastatic lung adenocarcinoma cells bearing CD74-ROS1 fusion interact with macrophages to promote their dissemination.

Approximately 40% of patients with lung adenocarcinoma (LUAD) often develop bone metastases during the course of their disease. However, scarcely any in vivo model of LUAD bone metastasis has been established, leading to a poor understanding of the mechanisms underlying LUAD bone metastasis. Here, we established a multiorgan metastasis model via the left ventricular injection of luciferase-labeled LUAD cells into nude mice and then screened out lung metastasis (LuM) and bone metastasis (BoM) cell subpopulations. BoM cells exhibited greater stemness and epithelial-mesenchymal transition (EMT) plasticity than LuM cells and initially colonized the bone and subsequently disseminated to distant organs after being reinjected into mice. Moreover, a CD74-ROS1 fusion mutation (C6; R34) was detected in BoM cells but not in LuM cells. Mechanistically, BoM cells bearing the CD74-ROS1 fusion highly secrete the C-C motif chemokine ligand 5 (CCL5) protein by activating STAT3 signaling, recruiting macrophages in tumor microenvironment and strongly inducing M2 polarization of macrophages. BoM cell-activated macrophages produce a high level of TGF-β1, thereby facilitating EMT and invasion of LUAD cells via TGF-β/SMAD2/3 signaling. Targeting the CD74-ROS1/CCL5 axis with Crizotinib (a ROS1 inhibitor) and Maraviroc (a CCL5 receptor inhibitor) in vivo strongly impeded bone metastasis and secondary metastasis of BoM cells. Our findings reveal the critical role of the CD74-ROS1/STAT3/CCL5 axis in the interaction between LUAD bone metastasis cells and macrophages for controlling LUAD cell dissemination, highlighting the significance of the bone microenvironment in LUAD bone metastasis and multiorgan secondary metastasis, and suggesting that targeting CD74-ROS1 and CCL5 is a promising therapeutic strategy for LUAD bone metastasis.

A consistency comparison between next-generation sequencing and the FISH method for gene rearrangement detection in B-cell lymphomas

Objective: To compare the consistency of lymphoma multigene detection panels based on next-generation sequencing (NGS) with FISH detection of B-cell lymphoma gene rearrangement. Methods: From January 2019 to May 2023, fusion genes detected by lymphoma-related 413 genes that targeted capture sequencing of 489 B-cell lymphoma tissues embedded in paraffin were collected from Henan Cancer Hospital, and the results were compared with simultaneous FISH detection of four break/fusion genes: BCL2, BCL6, MYC, and CCND1. Consistency was defined as both methods yielding positive or negative results for the same sample. The relationship between fusion mutation abundance in NGS and the positivity rate of cells in FISH was also analyzed. Results: Kappa consistency analysis revealed high consistency between NGS and FISH in detecting the four B-cell lymphoma-related gene rearrangement (P<0.001 for all) ; however, the detection rates of positive individuals differed for the four genes. Compared with FISH, NGS demonstrated a higher detection rate for BCL2 rearrangement, a lower detection rate for BCL6 and MYC rearrangement, and a similar detection rate for CCND1 rearrangement. No correlation was found between fusion mutation abundance in NGS and the positivity rate of cells in FISH. Conclusions: NGS and FISH detection of B-cell lymphoma gene rearrangement demonstrate overall good consistency. NGS is superior to FISH in detecting BCL2 rearrangement, inferior in detecting MYC rearrangement, and comparable in detecting CCND1 rearrangement.

Optimization of fusion detection in non-small cell lung cancer by amplicon-based RNA sequencing.

e20042 Background: In non-small cell lung cancer (NSCLC), more and more driver fusions are suitable for targeted therapy. Sequential or parallel DNA and RNA sequencing is an option, but it is costly and requires sufficiently high quality biopsy material. The purpose of this study was to confirm the consistency of fusion genes detected by DNA-based NGS and RNA-based NGS in the same sample, and to validate the performance of FFPE samples for RNA-based NGS. Methods: In this study, we evaluated the benefits of capture-based DNA sequencing and amplicon-based RNA sequencing in identifying gene fusion and exon-skipping events in 98 NSCLC patients. All cases were FFPE samples stored for 1-5 years. In addition, we investigated the limiting performance of RNA-based NGS for RNA initiation. Results: In the cases of positive DNA sequencing fusion samples, the fusion consistency detection rate of RNA sequencing was 95% (38/40). Two cases were not detected by RNA-based NGS, one was a five-year FFPE sample and the other was due to a probe not covered problem. In patients with negative DNA fusion genes, RNA sequencing could detect 12.07% more fusion-positive cases involving ALK (n = 1), ROS (n = 3), MET 14 exon skipping (n = 1), RET (n = 1), and BRAF (n = 1). Moreover, the FFPE samples stored for 3-5 years inevitably degrade the RNA, but could still be used for RNA-based sequencing. Among 66 FFPE samples stored for 3-5 years, the detection rate of fusion consistency between DNA sequencing fusion samples and RNA sequencing was 96.77%(30/31). In patients with negative DNA fusion genes, RNA sequencing was able to detect 14.29% (5/35) more fusion-positive cases. The performance verification of the other 8 samples with different RNA input amounts found that 87.5% (7/8) of the samples could meet the quality control of RNA sequencing and accurately detect fusion mutations as low as 1 ng. Conclusions: Molecular profiling based on mixed DNA and RNA sequencing improves the effectiveness of detection of fusion-driven NSCLC. The method was feasible on small tissue samples as well as FFPE samples stored for 3-5 years, and could greatly reduce the complexity and cost of molecular examination.

The combination of gemcitabine plus an anti-FGFR inhibitor can have a synergistic antitumor effect on FGF-activating cholangiocarcinoma

Anti-FGFR treatment for cholangiocarcinoma (CCA) with fibroblast growth factor receptor (FGFR) alteration is a promising treatment option. Since the antitumor mechanisms of anti-FGFR inhibitors and conventional cytotoxic drugs differ, synergistic effects can be possible. This study aimed to evaluate the efficacy of the combined administration of gemcitabine (GEM) and pemigatinib in CCA cells with FGFR2 alterations. To simulate the treatment for patients with 3 kinds of CCA, chemonaïve CCA with activation of the FGF pathway, chemo-resistant CCA with activation of the FGF pathway, and CCA without FGF pathway activation (as controls), we evaluated 3 different CCA cell lines, CCLP-1 (with a FGFR2 fusion mutation), CCLP-GR (GEM-resistant cells established from CCLP-1), and HuCCT1 (without FGFR mutations). There was no significant difference between CCLP-1 and HuCCT1 in GEM suspensibility (IC50 = 19.3, 22.6 mg/dl, p = 0.1187), and the drug sensitivity to pemigatinib did not differ between CCLP-1 and CCLP-GR (IC50 = 7.18,7.60 nM, p = 0.3089). Interestingly, only CCLP-1 showed a synergistic effect with combination therapy consisting of GEM plus pemigatinib in vitro and in vivo. In a comparison of the reaction to GEM exposure, only CCLP-1 cells showed an increase in the activation of downstream proteins in the FGF pathway, especially FRS2 and ERK. In association with this reaction, cell cycle and mitosis were increased with GEM exposure in CCLP-1, but HuCCT1/CCLP-GR did not show this reaction. Our results suggested that combination therapy with GEM plus pemigatinib is a promising treatment for chemonaïve patients with CCA with activation of the FGF pathway.

Afatinib Treatment for Advanced Mixed Non-small Cell Lung Cancer with CRISPLD2-NRG1 Fusion: A Case Report and Literature Review

Lung cancer is the most common malignant disease and the leading cause of cancer death in China. Non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancers, and the probability of NSCLC gene mutations is high, with a wide variety of types. With the development of next-generation sequencing (NGS) detection technology, more and more patients with rare fusion gene mutations are detected. Neuregulin 1 (NRG1) gene is a rare oncogenic driver that can lead to activation of human epidermal growth factor receptor 3 (Her3/ErbB3) mediated pathway, resulting in tumor formation. In this article, we reported a case of mixed NSCLC with CRISPLD2-NRG1 fusion detected by RNA-based NGS, who responsed to Afatinib well after 1 month of treatment, and magnetic resonance imaging (MRI) showed shrinkage of intracranial lesions. Meanwhile, we also compiled previously reported NSCLC patients with NRG1 rare gene fusion mutation, in order to provide effective references for clinical diagnosis and treatment. .

Abstract A005: Exploring liquid biopsy for the detection of bladder cancer: Unraveling RNA fusions

Abstract Introduction: This study delves into the promising realm of liquid biopsy for the non-invasive detection of bladder cancer. Leveraging urine as a valuable source, our investigation focuses on identifying RNA fusions associated with bladder cancer, with specific attention to ETV6-NTRK3 and PRCC-TFE3. The utilization of liquid biopsy offers a less invasive and more accessible approach to diagnosing bladder cancer, potentially revolutionizing the field of cancer diagnostics. These specific RNA fusions in urine samples may serve as reliable biomarkers for early detection and monitoring of bladder cancer. This research contributes to the ongoing efforts to enhance diagnostic methodologies to improve patient outcomes and streamline cancer management. Materials and Methods: Urine was collected from healthy male donors using UAS, a urine preservative from Novosanis, for improved urine preservation and stability. The urine samples with UAS were centrifuged at 16,000 xg for 10 min to obtain cf-urine. Three 20 mL aliquots of the precleared supernatant urine samples were spiked with an RNA fusion reference standard from Anchor Molecular containing ETV6-NTRK3 and PRCC-TFE3RNA at a concentration of 20ng/mL. All samples were extracted using the nRichDX Revolution Max20 cfTNA Isolation Kit. The eluants from the extracted samples were DNAse treated. The extracted cfTNAs profile was assessed on 4150 TapeStation System, Agilent, using RNA ScreenTape. Extracted nucleic acids from urine were evaluated using a RT-qPCR assay to quantify the number of copies of the fusion RNAs. Results: The Renal RNA fusions ETV6-NTRK3 and PRCC-TFE3RNA were successfully extracted using the nRichDX Revolution cfTNA Isolation Kit. TapeStation analysis demonstrated prominent 18S and 28S peaks, confirming the successful extraction of RNA from 20 mL urine samples. The mean percent recovery of the recovered RNA Fusion mutation was about 70% via RT-qPCR. Conclusion: Our findings indicate a recovery rate of approximately 70% for spiked RNA fusions in the urine samples. This research holds the promise of revolutionizing cancer diagnostics by introducing a method that is not only reliable but also convenient for early detection and monitoring. Identifying specific RNA fusions in urine samples as biomarkers is a significant step in enhancing diagnostic methodologies. As we contribute to these ongoing efforts, we aim to improve patient outcomes and streamline the management of bladder cancer through innovative and effective diagnostic approaches. Citation Format: Mayer Saidian, Jason Saenz, Carlos Hernandez, Lauren Lee, Andrew Dunnigan, Yabin Lu, Nafiseh Jafari. Exploring liquid biopsy for the detection of bladder cancer: Unraveling RNA fusions [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A005.

Mutation characteristics and molecular evolution of ovarian metastasis from gastric cancer and potential biomarkers for paclitaxel treatment

Ovarian metastasis is one of the major causes of treatment failure in patients with gastric cancer (GC). However, the genomic characteristics of ovarian metastasis in GC remain poorly understood. In this study, we enroll 74 GC patients with ovarian metastasis, with 64 having matched primary and metastatic samples. Here, we show a characterization of the mutation landscape of this disease, alongside an investigation into the molecular heterogeneity and pathway mutation enrichments between synchronous and metachronous metastasis. We classify patients into distinct clonal evolution patterns based on the distribution of mutations in paired samples. Notably, the parallel evolution group exhibits the most favorable prognosis. Additionally, by analyzing the differential response to chemotherapy, we identify potential biomarkers, including SALL4, CCDC105, and CLDN18, for predicting the efficacy of paclitaxel treatment. Furthermore, we validate that CLDN18 fusion mutations improve tumor response to paclitaxel treatment in GC with ovarian metastasis in vitro and vivo.

Meiotic drive against chromosome fusions in butterfly hybrids

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

Abstract 1958: EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs

Abstract RET fusions are an oncogenic driver mutation occurring in 1-2% non-small cell lung cancer (NSCLC). Currently, RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are approved for this patient population, demonstrating favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC. However, tumors eventually become refractory to selective RET inhibitor monotherapy. We sought to identify signaling pathways which mediate RET-independent acquired resistance to RET inhibitors and effective therapeutic combinations to overcome resistance. Using LC-2/ad (CCDC6-RET) NSCLC cells, we assessed the expression and activation of critical signaling pathways at a proteomic level following RET knockdown using reverse-phase protein array. RET knockdown resulted in rapidly increased adaptive upregulation of EGFR, HER2, MET, mTOR and MEK pathway signaling; observations were confirmed by Western Blotting. To determine whether EGFR or MET signaling facilitate RET inhibitor resistance, LC-2/ad and NCCE-TH1101 (KIF5B-RET) NSCLC cells were treated with increasing concentrations of selpercatinib or pralsetinib with or without ligands for these receptors, EGF or HGF respectively, and after five days cell viability was measured by CellTiter Glo. EGFR and MET signaling promoted RET TKI resistance in both cell lines. Next, to evaluate whether blockade of EGFR or MET signaling could enhance the activity of RET inhibitors, we treated LC-2/ad and NCCE-TH1101 cells with RET TKIs alone or in combination with the EGFR TKI erlotinib or poziotinib, or the MET inhibitor SU11274. We observed an additive effect with EGFR but not MET inhibitors when combined with RET TKIs. Western blot analysis revealed that EGFR signaling could reactivate mTOR and MAPK signaling after RET TKI treatment. Therefore, we next tested whether inhibition of these downstream pathways might enhance RET TKI sensitivity. We observed that treatment of LC-2/ad and NCCE-TH1101 cells with an mTOR inhibitor (everolimus) or a MEK inhibitor (trametinib) in combination with RET TKIs increased the anti-tumor activity or RET inhibitors. Finally, we established cells with acquired resistance to selpercatinib and pralsetinib. RET TKI resistant cells did not harbor secondary RET fusion mutations but underwent epithelial to mesenchymal transition and exhibited pan-RET inhibitor resistance. Moreover, RET TKI resistant cells showed increased cell surface expression of EGFR and MET by flow cytometry. RET TKI resistant cells were sensitive to mTOR or MEK inhibitors in combination with RET TKIs. Collectively, our findings indicate that in RET-fusion positive NSCLC tumor cells, activation of bypass pathways including the EGFR, MAPK, mTOR pathways may facilitate RET inhibitor resistance and that blockade of bypass pathways may enhance the efficacy of selective RET inhibitors and overcome acquired RET TKI resistance. Citation Format: Tomohiro Takehara, Monique B. Nilsson, Ximeng Liu, Hibiki Udagawa, Naoto Morikawa, Alissa Poteete, Junqin He, Xiaoxing Yu, Thiru Arumugam, Koichi Goto, John V. Heymach. EGFR and mTOR signaling facilitate RET-independent resistance to selective RET-TKIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1958.

Abstract 415: Keratin-fusion mutation promotes tumor aggressiveness via genomic instability induction

Abstract Keratin intermediate filament is one critical component of cellular architectures, which provides necessary mechanical support to vanquish environmental stresses. Our previous study shows that keratin fusion mutation promotes tumor aggressiveness via enhancing cancer stemness in oral squamous cell carcinoma. It implies proper functions of keratins play a critical role in maintain cell integrity and fate determination. In the present study, keratin fusion mutation functions as a driver to promote genomic instability via centrosome overduplication and trigger the formation of polyaneuploid cancer cell (PACC). Interestingly, genes involved in DNA damage repair were found upregulated in keratin fusion expressing cells, an important feature for mitotic slippage during cancer progression. In addition, PACCs caused by keratin fusion render cancer cells insensitive to cisplatin treatment, which coupled with lower levels of γH2AX induction and higher survival rates. Transcriptome analyses revealed enhanced activity for Regulation_of_Actin_Cytoskeleton in the PACCs, leading to increased actin filament network and higher invasiveness. In summary, keratin fusion mutation promote cancer aggressiveness through genomic instability-mediated PACC formation and cytoskeleton remodeling, which lead to enhanced drug resistance and metastasis. Citation Format: Hsiang-Hao Chuang, Brian Yu-Ting Kuo, Kalpana Sriramadasu, Yi-Che Chen, Jim Jinn-Chyuan Sheu. Keratin-fusion mutation promotes tumor aggressiveness via genomic instability induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 415.

THE GENOMIC LANDSCAPE OF THE SWITCH/SUCROSE NON-FERMENTABLE CHROMATIN REMODELING COMPLEX IN ACUTE MYLEOID LEUKEMIA

The SWI/SNF chromatin remodeling complex is involved in the regulation of gene expression required for processes such as cell maintenance and differentiation in hematopoietic stem cells. Abnormalities in the SWI/SNF subunits involved in the homeostasis of hematologic processes contribute to the initiation or progression of hematologic malignancies, but the mechanisms underlying this phenotype are not yet fully understood. The aim of study is to comprehensively identify mutations and expression profiles in the genes forming the SWI/SNF complex using bioinformatics tools, with a focus on understanding the underlying mechanisms. Genomic sequences and expression profiles of an AML cohort (n:872) were obtained from using tools and subsequently analyzed. PolyPhen-2, SIFT, and Mutation Assessor tools were used to estimate the oncogenic/pathogenic effects of mutations identified in 9 genes encoding subunits of the complex ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCE1, SMARCB1, DPF2, PMBR1, and BCL7A in AML pathogenesis. STRING analysis was performed to better understand the functional relationships of the mutant proteins in cellular processes. Furthermore, to the mutation profile, gene expression and survival profiles were also determined. A total of 17 genetic abnormalities were determined in 9 genes, including 9 missense, 6 frameshift mutations, 1 mutation in the splice region, and 1 fusion mutation. In the AML cohort, the expression levels of ARID1A, ARID1B, SMARCA2, and PMBR1 were significantly higher in the patient group compared to the healthy group (p