BRAF Fusion Research Articles

LGG-50. POOR RESPONSE TO MEKI RE-INITIATION IN PEDIATRIC LOW-GRADE GLIOMA AFTER TREATMENT DISCONTINUATION. A PLGG TASKFORCE STUDY

Abstract BACKGROUND Pediatric low-grade gliomas (pLGG) exhibit dramatic objective response to treatment with MEKi (Mitogen-Activated Protein Kinase inhibitors). Although the initial response to MEKi is promising, it is unknown whether it is safe to stop MEKi therapy and if the response to re-initiating MEKi would have the same impact. The study objective was to compare the first and second response to MEKi in patients with pLGG who stopped therapy and had further tumor progression. METHODS PLGG identified at neurooncology centers in Canada, Australia, and US, which were treated twice with MEKi were included. Second therapy was defined as MEKi upon progression for tumors where treatment was stopped. Response to MEKi between 2 treatments (MEKi1, MEKi2) were analyzed by using RANO criteria. RESULTS Mature data on 19 patients reveals a median patient age of 13 years (range 7–24), and the most common tumor location is hypothalamic/chiasmatic (89%). PLGG alterations include KIAA1549:BRAF fusion in 69%, NF1 in 21%, and other alterations in 10%. The overall response (minor and partial) to MEKi1 was 63% compared to 26% in MEKi2 (p=0.04). Furthermore, upon restarting MEKi2, 11% had further growth on therapy in contrast to none at MEKi1. MEKi1 resulted in average of 37% size decrease (range, 3%-98%), including 6 PLGG with over 50% response. In contrast, the average decrease to MEKi2 was 10% (range, 6%-40%, p=0.005), and none had >50% size reduction. Comparing each patient MEKi1 to MEKi2 revealed that initial response was superior to the second one in all PLGG. CONCLUSION These data suggest that the second response to MEKi is inferior when compared to initial therapy. Expansion of this cohort and biomarker correlates are ongoing. Biological insights are required for this observation, and careful consideration should be made for stopping MEKi, especially in strategic locations such as the optic pathway.

MODL-08. PATIENT-DERIVED ORGANOIDS RECAPITULATE THE MOLECULAR LANDSCAPE OF PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Pediatric brain tumors (PBT) are the leading cause of non-accidental death in pediatric age. These tumors are rare and lack adequate experimental models capable of accurately capturing their complex molecular landscape. METHODS AND RESULTS Our team has established specific protocols for the generation and expansion of ex-vivo patient-derived organoids (PDO) from fresh surgical material of a variety of PBT, within a clinically relevant timeframe. These include pilocytic astrocytomas, a pleomorphic xanthoastrocytoma, a rosette-forming glioneuronal tumor, a supratentorial ependymoma, diffuse high-grade gliomas, a medulloblastoma, an atypical teratoid rhabdoid tumor, and a meningioma. The PDO cultures were set within 7-15 days, with organoids reaching 200-800 μm in diameter. Immunofluorescence analysis using confocal high-content microscopy imaging revealed that PDO kept the protein markers typically found in the corresponding primary tumors. Likewise, at a genomic level, PDO recapitulated the molecular landscape of corresponding primary tumors, as denoted by the maintenance of NF1, PIK3CA, and FGFR1mutations (rosette-forming glioneuronal tumor), KIAA1549::BRAF fusions (pilocytic astrocytomas), ZFTA::RELA fusion(supratentorial ependymoma) or gene amplifications, such as KRAS, CDK4, MDM2, GLI1, PTPN11 and PI3KCA. Additionally, PDO exhibited minimal genetic drift after over one month in culture. In three PBT cases from a medulloblastoma, an atypical teratoid rhabdoid tumor, and a meningioma, where no molecular alterations were detected using a comprehensive NGS panel, we are running DNA methylation arrays and copy number variations. CONCLUSIONS Our results underscore the potential of PDO as a robust ex-vivo experimental model. The generated PDO cultures can be rapidly established and retain key molecular features of corresponding primary tumors. This preclinical model presents versatile applications, serving as a valuable molecular tool for precision medicine approaches in PBT patients, the study of PBT biology, and the acceleration of drug development processes within the context of PBT.

LGG-29. BEYOND MAPK SIGNALING - GENOME-SCALE CRISPR/CAS9 SCREENS REVEAL NEW TARGETS FOR TREATMENT OF KIAA1549::BRAF-DRIVEN PEDIATRIC LOW-GRADE GLIOMA

Abstract Since the KIAA1549::BRAF fusion was discovered as the most common driver of pediatric low-grade glioma (pLGG), it has been hypothesized that the fusion induces oncogenicity through BRAF activation as a result of KIAA1549 replacing the BRAF negative regulatory N-terminus. This led to the rapid translation of MAPK pathway inhibitors into the clinical setting. Despite most tumors exhibiting promising initial responses, some tumors are not sensitive and about half of responsive tumors grow back after treatment cessation. Therefore, strategies that result in sustained tumor responses are desperately needed. We have recently performed genome-scale CRISPR/Cas9 screens across isogenic neural stem cell models transduced to express pLGG-associated oncogenes (KIAA1549::BRAF, BRAFV600E, and multiple FGFR1 and MYB family alterations) to generate a dependency map of genetic vulnerabilities associated with expression of these oncogenes. We also included normal neural stem cells to allow identification of genetic dependencies specifically induced by the expression of each oncogene. Surprisingly, through these efforts, we have discovered KIAA1549::BRAF expressing cells to harbor striking and specific dependency on multiple members of an enzymatic complex that exerts its activity outside of the MAPK signaling axis*. Interestingly, this enzymatic complex has been described to modify only a few substrates, including KIAA1549, suggesting specificity. We have now validated this dependency across other isogenic models of KIAA1549::BRAF-expressing cells. Finally, our KIAA1549::BRAF-expressing models exhibit preferential sensitivity to a tool compound that targets our novel enzyme, suggesting novel therapeutic potential for KIAA1549::BRAF. These findings highlight a MAPK pathway-independent avenue for therapeutically targeting the most frequent genomic alterations in pediatric brain tumors. Consequently, we also propose that the fusion partner KIAA1549 is instrumental for the aberrant BRAF signaling driving pLGGs. (* we are currently in the process of working with our IP offices to ensure that we can disclose the names of the genes and proteins at the ISPNO meeting)

LGG-20. SELUMETINIB IN PEDIATRIC PATIENTS WITH RELAPSED/REFRACTORY OPTIC PATHWAY/HYPOTALAMIC LOW GRADE GLIOMA: A MONOINSTITUTIONAL EXPERIENCE

Abstract BACKGROUND Pediatric low-grade gliomas (pLGG) are the most common central nervous system tumors in children. The optic pathway and hypothalamic (OPH) frequent localizations are correlated with multiply relapses due to inoperability, thus emphasizing the need for alternative treatments. The results of activation of the BRAF oncogene through a tandem duplication resulting in a KIAA1549–BRAF fusion is the most common genetic aberration. Selumetinib is an oral mitogen-activated protein kinase (MEK) 1/2 inhibitor with promising efficacy and good tolerability on relapsed/refractory pLGG. METHODS From January 2021 to January 2024 three patients with refractory OPH-pLGG with KIIA1549-BRAF fusion were treated with Selumetinib 25 mg/sqm/day (compassionate use) all after two lines of chemotherapy. They were males, mean age at diagnosis 8 months, at starting Selumetinib 74 months. All had partial surgery at diagnosis and two of them other debulking surgeries during the treatment. All patients had numerous events of hydrocefalous with need of shunt revision. RESULTS Two patients experienced grade-3 stomatitis, one grade 3 nausea-vomiting, one grade 3 gastritis, one grade 1 increased level of CPK (CTCAE-V5.0). For grade 3 toxicity Selumetinib had to be stopped for a maximum of 7 days with clinical benefit. Two of them have completed treatment with Selumetinib, one is ongoing after 20 months, all with radiological stable disease at MRI. Median time of treatment is 26 months (range 20-30 months). All three experienced a trend in reduction of emergency room admission for ventricular-shunt problems. Median PFS at starting Selumetinib was 62 months, median OS is 97 months. CONCLUSIONS Selumetinib has a good profile of toxicity, allowing a good quality of life procrastinating radiotherapy and reducing surgery events in a context of heavily pretreated relapsed/refractory patients. Further data are needed on both visual and auxological parameters and late side effects.

IMG-05. USING CONVOLUTIONAL NEURAL NETWORKS TO IDENTIFY COMMON MOLECULAR ALTERATIONS IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Artificial intelligence (AI) and deep learning have had increased uses in healthcare. Initial studies have shown it is possible to predict multiple tumor types in adults and children using convolutional neural networks (CNN), a form of deep learning, using MRIs. In pediatric CNS tumors, molecular alterations are used in both diagnosis and treatment decisions. This project aims to verify that we can use deep learning to help diagnose and molecularly classify primary pediatric CNS tumors. Previously, we used Recurrent Health’s platform to predict high vs. low grade CNS lesions to >95% recall and 85% precision in pediatric patients. The objective of this study is to validate the AI’s ability to predict common genetic alterations with 90% accuracy. METHODS This retrospective study evaluated patients (ages 0-21) with histologically confirmed primary CNS tumors that underwent BRAF molecular analysis. We collected demographic data: age, tumor location, and presence of BRAF mutations/fusions. The data was divided into a training and validation set (80%/20% split). To determine the viability of models, we will measure specificity, sensitivity, and F1 Score (Sorenson-Dice coefficient) for predicting genetic alterations. Model score confidence was achieved through K-fold cross-validation and statistical power analysis with a finite population coefficient to determine the appropriate sample size. RESULTS Our retrospective test included 136 patients with multiple scans for training, model validation and testing. The CNN was able to predict presence of BRAF fusions with >95% recall/sensitivity, >76% precision/specificity and F1/accuracy >87% while predicting BRAFV600 mutations with >75% recall/sensitivity, >95% precision/specificity and F1/accuracy >85%. These metrics indicate strong discrimination in classification. CONCLUSIONS Preliminary data suggests the accuracy of the model in identifying the presence of BRAF mutations, providing a potential opportunity for a non-invasive tool to help aid in diagnosis and help guide initiation of targeted therapy.

LGG-26. A ROLE FOR BRAF FUSION PARTNERS IN ONCOGENIC SIGNALING

Abstract t was originally hypothesized that loss of BRAF N-terminal negative regulatory domains, which are deleted in the KIAA1549:BRAF rearrangement, results in constitutive activation of the BRAF kinase. This model suggests that BRAF fusion partners are dispensable for BRAF oncogenic signaling. Paradoxically, our data suggest expression of truncated BRAF is insufficient for transformation and identify specific domains in KIAA1549 that are necessary for transformation. These domains are critical for regulating subcellular localization of the fusion protein and highlight an aberrant pattern of cellular localization that is not observed with wildtype BRAF. Altered subcellular localization results in proteolytic cleavage of KIAA1549:BRAF, and we identify the protease responsible for this cleavage event. This protease is therapeutically tractable and clinically relevant inhibitors have been developed, presenting a MAPK-independent mechanism to target the fusion. We also highlight an unexpected role for rare fusion partners, including FAM131B, in BRAF activation. In total, these data suggest that BRAF fusion partners are not indispensable for transformation as was previously thought, presenting unexplored opportunities to therapeutically target pLGG tumors with BRAF rearrangements. We are currently working on the IP measures required to disclose the specific enzyme(s) at ISPNO.

METB-11. HOW METHYLATION PROFILING HELPS IN DIAGNOSING AND TREATING PEDIATRIC BRAIN TUMORS IN INDONESIA

Abstract BACKGROUNDS Pediatric brain tumors (PBT) exhibit extensive heterogeneity, impacting treatment outcomes. While traditionally classified based on histopathological features, recent WHO classifications have integrated DNA methylation array profiling amongst other molecular characteristics, which improves diagnostic accuracy and thus treatment decisions. However, access to this technology is limited in low- and middle-income countries (LMICs) like Indonesia. As part of the MNP Outreach Consortium, we aimed to introduce methylation profiling in Indonesia to aid in diagnosing and stratifying PBT patients. METHODS In 2023, we conducted a case series involving seven challenging PBT cases from Cipto Mangunkusumo National General Hospital, re-assessing them with methylation profiling. RESULTS Through collaboration with the Institute of Neuropathology Heidelberg and the DKFZ, our centre adopted a discerning approach to re-evaluate histopathological samples from complex cases. This endeavour yielded notable results, reaffirming four initial diagnoses and altering three others Amongst the findings, one case initially diagnosed as anaplastic ependymoma, CNS WHO grade 3, was reclassified as a supratentorial ependymoma with YAP1 fusion gene, based on methylation class. Noteworthy within this latter group, one case initially categorized as low-grade pilocytic astrocytoma was reclassified as high-grade diffuse midline glioma, H3 K27 altered. Moreover identifying the copy number plot could indicate a KIAA1549::BRAF fusion to reclassify a case from diffuse pediatric glioma WHO grade 2 to pilocytic astrocytoma WHO grade 1. The recurrent pigmented neuroectodermal tumor of infancy (MNTI) was reclassified as Medulloblastoma, non-WNT/non-SHH, group 3 subtype II on methylation profiling, highlighting molecular similarities and potential treatment insights. These findings offer insights into genetic mechanisms and guide treatment strategies. CONCLUSION Accurate diagnosis, facilitated by advanced molecular profiling, enhances prognostication and enables personalized treatment plans, even in LMICs. This underscores the importance of expanding access to advanced diagnostic technologies in resource-limited settings.

Molecular-targeted therapy for childhood low-grade glial and glioneuronal tumors.

Since the discovery of the association between BRAF mutations and fusions in the development of childhood low-grade gliomas and the subsequent recognition that most childhood low-grade glial and glioneuronal tumors have aberrant signaling through the RAS/RAF/MAP kinase pathway, there has been a dramatic change in how these tumors are conceptualized. Many of the fusions and mutations present in these tumors are associated with molecular targets, which have agents in development or already in clinical use. Various agents, including MEK inhibitors, BRAF inhibitors, MTOR inhibitors and, in small subsets of patients NTRK inhibitors, have been used successfully to treat children with recurrent disease, after failure of conventional approaches such as surgery or chemotherapy. The relative benefits of chemotherapy as compared to molecular-targeted therapy for children with newly diagnosed gliomas and neuroglial tumors are under study. Already the combination of an MEK inhibitor and a BRAF inhibitor has been shown superior to conventional chemotherapy (carboplatin and vincristine) in newly diagnosed children with BRAF-V600E mutated low-grade gliomas and neuroglial tumors. However, the long-term effects of such molecular-targeted treatment are unknown. The potential use of molecular-targeted therapy in early treatment has made it mandatory that the molecular make-up of the majority of low-grade glial and glioneuronal tumors is known before initiation of therapy. The primary exception to this rule is in children with neurofibromatosis type 1 who, by definition, have NF1 loss; however, even in this population, gliomas arising in late childhood and adolescence or those not responding to conventional treatment may be candidates for biopsy, especially before entry on molecular-targeted therapy trials.

Tovorafenib (Ojemda) for pediatric low-grade glioma.

Tovorafenib (Ojemda – Day One), a type II RAF kinase inhibitor, has received accelerated approval from the FDA for treatment of patients ≥6 months old with relapsed or refractory pediatric low-grade glioma harboring a BRAF fusion or rearrangement or a BRAF V600 mutation. Tovorafenib is the first systemic treatment to be approved in the US for pediatric low-grade gliomas with BRAF fusions. Accelerated approval of tovorafenib was based on response rates and duration of response.

Implementing personalized lung cancer care with spatial profiling, patient derived organoids, and rationally designed drug testing.

e20521 Background: Lung cancer (LC) remains the leading cause of cancer-related death in 2024, with most LC diagnoses with non-small cell lung cancer (NSCLC) subtype. Tyrosine kinase inhibitors (TKI) and targeted therapy against oncogenic drivers (KRAS, EGFR, or ALK fusions, others) are effective initial therapies; however, nearly all treated patients eventually develop resistance to therapy. Key reasons are the focus on limited predictive biomarkers and lack of patient derived LC models that reflect the underlying heterogeneity and the various phenotypic cell states among subclones within and between patients. Methods: We report the establishment of a novel platform for personalized LC care by refining the resolution of molecular interrogation of diagnostic and resistance biopsies to the single cell level, and with spatial profiling using single cell spatial multiomic (scSpMO) and coupling these enhanced diagnostic tools with functional validation with rational designed drug screens in patient-derived organoids (PDOs). Results: For deriving LC PDOs in epithelial and tumor microenvironment (TME) conditions to faithfully maintain the histological and genetic features of their respective LC tissues, we developed enrichment conditions for LC PDOs guided by tumor mutation variants and activated pathways. PDOs are maintained under the same treatment condition in the clinic (e.g., Lorlatinib) and drug testing is tailored to identify potentially most effective next lines of therapy. With nine patients enrolled and others in ongoing studies, single cell PDOs (at > 80% establishment rate) were used to determine activated pathways, lineage plasticity and acquired resistance. Functional assays for acquired resistance such as PI3K/AKT signaling, Src kinase, BRAF fusion and MET hyperactivity allow the identification of potentially novel lines of therapy and treatment sequence and/or combinations for each patient, through the compassionate care program and/or trial participation. Conclusions: Our platform when empowered by combining datasets from scRNA-seq and scSpMO signatures, with validated functional drug responses in PDOs, offers the ability to perform high-content assays, predict and/or act on resistance to therapy for each patient, and provides a path for precision medicine-guided impact on patient care.

Midline Low-Grade Gliomas of Early Childhood: Focus on Targeted Therapies.

Midline low-grade gliomas (mLGGs) of early childhood have a poorer prognosis compared with tumors of other localizations and in older patients. LGGs are associated with aberrant activation of RAS-RAF-MEK pathway, and pharmacological inhibition of the pathway has therapeutic promise. The aim of this study was clinical and molecular characterization of infantile mLGGs, with emphasis on the efficacy of targeted kinase inhibition. This study enrolled 40 patients with mLGG age <3 years. The majority of the patients (30/40) received first-line chemotherapy (CT) as per International Society of Paediatric Oncology LGG 2004 guidelines. In all patients, molecular genetic investigation of tumor tissue by polymerase chain reaction and RNA sequencing was performed. The median follow-up was 3.5 years. First-line CT failed in 24 of 30 recipients. The identified molecular profiles included KIAA1549::BRAF fusions in 26 patients, BRAF V600E in six patients, FGFR1::TACC1 fusions in two patients, and rare fusion transcripts in four patients. At disease progression, targeted therapy (TT) was initiated in 27 patients (22 patients received trametinib) on the basis of molecular findings. TT was administered for a median of 16 months, with partial response achieved in 12 of 26 (46%) patients in which response was evaluated. Severe adverse events were detected only on trametinib monotherapy: acute damage of GI or urinary mucosa complicated by hemorrhage and development of transfusion-dependent anemia in four patients and grade 3 skin toxicity in three patients. mLGGs of early childhood are often aggressive tumors, resistant to CT, and frequently require alternative treatment. The majority of patients harbor druggable molecular targets and respond to molecular TT.

A phase 1 study of the BET inhibitor ZEN003694 in combination with the MEK1/2 inhibitor binimetinib in solid tumors with RAS pathway alterations and triple-negative breast cancer (NCI number 10449).

TPS3182 Background: Bromodomain and extra-terminal (BET) proteins serve as epigenetic readers and regulate transcription by binding acetylated lysines in histones. BET inhibitors target this epigenetic machinery modifying the expression of oncogenes and have the potential to overcome drug resistance in different settings. Resistance to mitogen-activated extracellular kinase (MEK) inhibition appears to rely upon changes in gene expression, a process that can be potentially inhibited via BET blockade. Preclinical data have demonstrated synergistic efficacy of dual MEK and BET inhibition in MAPK-altered solid tumors. Triple negative breast cancer (TNBC) is a disease frequently found to have gene amplifications in BRAF, NF1 mutations, and upstream regulators of the MAPK pathway and preclinical work has shown efficacy with dual inhibition of MEK and BET. Methods: This phase 1, open-label, multicenter study was designed to evaluate the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the BET inhibitor ZEN003694 in combination with the MEK inhibitor binimetinib in solid tumors with RAS pathway alterations and TNBC. The study has two parts: dose escalation (part 1) which plans to recruit a maximum of 30 patients and dose expansion (part 2), which will comprise 12 patients. The primary objective of part 1 is to determine the MTD and recommended Phase 2 dose (RP2D), whereas the primary objective of dose expansion is to evaluate safety and toxicity. Secondary objectives include tolerability, pharmacokinetics (PK), and antitumor activity. Additional analyses will explore epigenetic changes such as ChIPseq to determine levels of H3K27ac and ATACseq to determine treatment-induced changes in open chromatin regions. ZEN003694 is given orally daily, and binimetinib is administered orally twice daily in 28-day cycles. Dose escalation is being conducted using a standard 3+3 cohort design to determine the MTD. Part 1 requires evaluable or measurable disease, whereas Part 2 requires measurable disease as per RECIST v1.1. Patients must have an ECOG performance status of ≤2. Included tumors are TNBC (estrogen receptor ≤1%, progesterone receptor ≤1%, human epidermal growth factor receptor 2 (HER2) 0–1+ or non-amplified) or any other solid tumor with actionable MAPK alterations (e.g. KRAS, NRAS, HRAS, or BRAF activating mutations, inactivating NF1 mutations, or BRAF fusions). Patients with any PI3K pathway activating genomic alterations are excluded. Prior therapy with BET, RAF, MEK, or ERK inhibitor is not permitted. This study is currently ongoing in dose escalation. Clinical trial information: NCI number 10449.

Phase II Study of Ulixertinib in Children and Young Adults With Tumors Harboring Activating Mitogen-Activated Protein Kinase Pathway Alterations: APEC1621J of the National Cancer Institute-Children's Oncology Group Pediatric MATCH Trial.

The National Cancer Institute-Children's Oncology Group (NCI-COG) Pediatric MATCH trial assigns patients age 1-21 years with refractory malignancies to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with activating alterations in the mitogen-activated protein kinase pathway were treated with ulixertinib, an extracellular signal-regulated kinase (ERK)1/2 inhibitor. As there were no previous pediatric data, ulixertinib was initially tested in a dose escalation cohort to establish the recommended phase II dose (RP2D) before proceeding to the phase II cohort. Ulixertinib was administered at 260 mg/m2/dose orally twice a day (dose level 1 [DL1], n = 15) or 350 mg/m2/dose orally twice a day (DL2, n = 5). The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival (PFS). Twenty patients (median 12 years; range, 5-20) were treated, all evaluable for response. CNS tumors comprised 55% (11/20) of diagnoses, with high-grade glioma and low-grade glioma most common (n = 5 each). All CNS tumors except one harbored BRAF fusions or V600E mutations. Rhabdomyosarcoma (n = 5) was the most frequent non-CNS diagnosis. DL1 was declared the RP2D in the dose escalation cohort after dose-limiting toxicities in Cycle 1 occurred in 1/6 patients at DL1 and 2/5 patients at DL2, including fatigue, anorexia, rash, nausea, vomiting, diarrhea, dehydration, hypoalbuminemia, and hypernatremia. No objective responses were observed. Six-month PFS was 37% (95% CI, 17 to 58). Three patients with BRAF-altered CNS tumors achieved stable disease >6 months. Ulixertinib, a novel targeted agent with no previous pediatric data, was successfully evaluated in a national precision medicine basket trial. The pediatric RP2D of ulixertinib is 260 mg/m2/dose orally twice a day. Limited single-agent efficacy was observed in a biomarker-selected cohort of refractory pediatric tumors.

Paediatric pancreatic acinar cell carcinoma with a novel SEC31A-BRAF fusion gene.

Paediatric pancreatic acinar cell carcinoma (PACC) presents a diagnostic challenge, often confused with pancreatoblastoma (PB) due to its rarity. It is crucial to differentiate between PB and PACC, given their distinct therapeutic strategies and prognoses. Histologically, the absence of squamoid nests and scarcity of tumor mesenchyme support PACC. Conversely, the identification of a BRAF alteration leans towards PACC. Here, we present the case of an 8-year-old girl with a well-defined mass in the pancreas. The tumor exhibited a SEC31A-BRAF fusion gene and amplification of 18p, showcasing unequivocal acinar differentiation and a minor degree of neuroendocrine differentiation. Additionally, the tumor displayed scant fibrous stroma, and an absence of squamoid nests, further supporting PACC. Notably, this is the first reported instance of a solid tumor featuring a SEC31A-BRAF gene fusion. The discovery of this novel fusion gene expands our understanding of BRAF fusion partner profiles, particularly in the context of paediatric PACC.

Multifocal Papillary Thyroid Carcinomas With Discordant Molecular Drivers: Emphasizing the Morphology and Collision Tumors.

Multifocal papillary thyroid carcinomas (PTCs) are common and the majority of the tumors harbor mutual BRAF p.V600E mutation. This study aimed to investigate a contemporary series of multifocal PTCs with discordant molecular drivers. Consecutive thyroidectomies diagnosed with multifocal PTCs ≥0.5cm between 2019 and 2023 were reviewed. Immunohistochemistry (IHC) for BRAF VE1 was performed for all tumors. Cases with discordant BRAF IHC results or morphologic discrepancy were identified, and BRAF IHC-negative tumors were subjected to RAS Q61R IHC and/or targeted RNA next-generation sequencing. A total of 770 patients with a main PTC ≥0.5cm were identified; 255 (33.1%) had multifocal disease, and 142 (18.4%) had at least another PTC ≥0.5cm. Among them, 13 cases (9.2%, 13/142) had discordant molecular drivers. Twelve cases had one or more BRAF-positive PTCs accompanied by a BRAF-negative PTC (3 with CCDC6::RET fusion, 1 with NCOA4::RET fusion, 1 with ACBD5::RET fusion, 2 with ETV6::NTRK3 fusion, 1 with TG::FGFR1 fusion, 1 with LMTK2::BRAF fusion, 1 with AGK::BRAF fusion and RAS p.Q61R mutation, 1 with RAS p.Q61R mutation, and 1 without detectable molecular drivers). The last case had tumors with discordant fusion drivers (VIM::NTRK3 and TNS1::BRAF). Most cases showed tumors that were morphologically distinct (92.3%, 12/13) and occurred in the contralateral lobes (76.9%, 10/13). Notably, we identified 4 cases (30.8%) that presented as collision tumors and 6 cases (46.2%) that showed lymph node metastases, including 2 with simultaneous involvement by tumors with discordant molecular drivers, as novel findings. In summary, a subset (9.2%) of multifocal PTCs had discordant molecular drivers and 84.6% of them were a combination of BRAF-positive and kinase gene fusion-associated PTCs, most with distinct morphologies. Almost half of the cases had nodal metastasis and a third of them showed simultaneous involvement by tumors with discordant molecular drivers. The results highlight the clinical importance of identifying such cases, given the potentially different treatments.

Genetic Landscape and Its Prognostic Impact in Children With Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare myeloid neoplasm that predominantly affects young children. To investigate genetic alterations and their correlation with clinical characteristics and prognosis in pediatric LCH. We performed targeted sequencing to detect mutations in LCH lesions from pediatric patients. A total of 30 genomic alterations in 5 genes of the MAPK pathway were identified in 187 of 223 patients (83.9%). BRAF V600E (B-Raf proto-oncogene, serine/threonine kinase) was the most common mutation (51.6%), followed by MAP2K1 (mitogen-activated protein kinase kinase 1) alterations (17.0%) and other BRAF mutations (13.0%). ARAF (A-Raf proto-oncogene, serine/threonine kinase) and KRAS (KRAS proto-oncogene, GTPase) mutations were relatively rare (2.2% and 0.9%, respectively). Additionally, FNBP1 (formin-binding protein 1)::BRAF fusion and MAP3K10 (mitogen-activated protein kinase kinase 10) mutations A17T and R823C were identified in 1 case each, with possible constitutive activation of ERK1/2 phosphorylation. BRAF V600E was more frequent in patients with risk organ involvement, while MAP2K1 mutation was more prevalent in patients with single-system LCH (P = .001). BRAF V600E was associated with craniofacial bone, skin, liver, spleen, and ear involvement (all P < .05). Patients with other BRAF mutations had a higher proportion of spinal column involvement (P = .006). Univariate analysis showed a significant difference in progression-free survival among the 4 molecular subgroups for patients treated with first-line therapy (P = .02). According to multivariate analysis, risk organ involvement was the strongest independent adverse prognostic factor (hazard ratio, 8.854; P < .001); BRAF or MAP2K1 mutation was not an independent prognostic factor. Most pediatric patients with LCH carry somatic mutations involving the MAPK pathway, correlating with clinical characteristics and outcomes for first-line chemotherapy.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Frequency of Common and Uncommon BRAF Alterations among Colorectal and Non-Colorectal Gastrointestinal Malignancies.

The predictive and prognostic role of BRAF alterations has been evaluated in colorectal cancer (CRC); however, BRAF alterations have not been fully characterized in non-CRC gastrointestinal (GI) malignancies. In the present study, we report the frequency and spectrum of BRAF alterations among patients with non-CRC GI malignancies. Patients with CRC and non-CRC GI malignancies who underwent somatic tumor profiling via a tissue-based or liquid-based assay were included in this study. Gain-of-function BRAF alterations were defined as pathogenic/likely pathogenic somatic short variants (SVs), copy number amplifications ≥8, or fusions (RNA or DNA). Among 51,560 patients with somatic profiling, 40% had CRC and 60% had non-CRC GI malignancies. BRAF GOF alterations were seen more frequently in CRC (8.9%) compared to non-CRC GI malignancies (2.2%) (p < 0.001). Non-CRC GI malignancies with the highest prevalence of BRAF GOF alterations were bile duct cancers (4.1%) and small intestine cancers (4.0%). Among BRAF GOF alterations, class II (28% vs. 6.8%, p < 0.001) and class III (23% vs. 14%, p < 0.001) were more common in non-CRC GI malignancies. Among class II alterations, rates of BRAF amplifications (3.1% vs. 0.3%, p < 0.001) and BRAF fusions (12% vs. 2.2%, p < 0.001) were higher in non-CRC GI malignancies compared to CRC. Non-CRC GI malignancies demonstrate a distinct BRAF alteration profile compared to CRC, with a higher frequency of class II and III mutations, and more specifically, a higher incidence of BRAF fusions. Future studies should evaluate clinical implications for the management of non-CRC GI patients with BRAF alterations, especially BRAF fusions.

Columnar Cell Thyroid Carcinoma: A Heterogeneous Entity Demonstrating Overlap Between Papillary Thyroid Carcinoma and Follicular Neoplasms.

Columnar cell papillary thyroid carcinoma (CC-PTC) is a morphologic subtype of papillary thyroid carcinoma with a variable prognosis. It is characterized by neoplastic thyroid follicular-derived cells with pseudostratified columnar morphology arranged in papillary or follicular structures with supranuclear or subnuclear vacuoles. The molecular profile of this subtype has only recently come under scrutiny, with mixed results. The aim of this study is to further explore the morphologic, immunohistochemical, and genetic profile of CC-PTC, as well as to correlate these features with clinical outcomes. CC-PTC cases were identified from 3 institutions. Immunohistochemistry (ER, CDX2) and molecular testing (DNA and RNA sequencing) were performed. Clinicopathologic parameters and patient outcomes were recorded. Twelve cases (2006-2023) were identified, all in adults (age 45-91). Two presented with disease outside the thyroid gland (neck and mediastinum) and two presented with distant metastasis. Four were high-grade differentiated thyroid carcinomas (necrosis or mitoses), one of which died of disease. Four were noninvasive or minimally invasive, one of which locally recurred. Three patients had lymph node metastases. ER and CDX2 were positive in 73% and 50%, respectively. Pathogenic mutations were found in TERT promoter (n = 3), RAS (n = 2), ATM, NOTCH1, APC, and ESR1, along with cases bearing AGK::BRAF fusion (n = 1), BRAF VE1 expression (n = 1), and NF2 loss (n = 1). This study represents the largest molecularly defined cohort of non-oncocytic thyroid carcinomas with columnar cell morphology. These tumors represent a genetically and behaviorally heterogeneous group of neoplasms, some of which have RAS-like or follicular neoplasm-like genetics, some of which have BRAF-p.V600E-like or classic papillary thyroid carcinoma-like genetics, and some of which remain unclear. Noninvasive or minimally invasive tumors showed an indolent course compared to those with angioinvasion, gross extrathyroidal growth, or high-grade morphology. Consideration could be given to reclassification of this neoplasm outside of the subtyping of papillary thyroid carcinoma in light of its genetic diversity, distinct morphology, and clinical behavior more closely aligned with follicular thyroid neoplasms.

A case of hyalinizing trabecular tumor of the thyroid: diagnostic significance of PAX8-GLIS3 fusion

BackgroundHyalinizing trabecular tumor (HTT) is an uncommon follicular cell-derived thyroid tumor classified as a low-risk neoplasm by the World Health Organization Classification of Tumors of Endocrine Organs, 5th edition. The PAX8-GLIS3 gene fusion is reportedly a pathognomonic genetic alteration of HTT.Case presentationA 43-year-old Japanese female was incidentally discovered to have an 8-mm, well-defined, hypoechoic mass in the left lobe of the thyroid gland by ultrasound examination. Contrast-enhanced computed tomography scan revealed a solid mass exhibiting slight homogeneous enhancement in the lower pole of the thyroid gland. The mass was diagnosed as atypia of undetermined significance by fine-needle aspiration cytology. The patient underwent left hemithyroidectomy with routine central compartment dissection. Histologic findings revealed tumor cells with elongated nuclei and intranuclear pseudoinclusions arranged with trabeculae architecture or small nests in hyalinized stroma. Weak membranous and cytoplasmic staining was found by MIB1 (Ki-67) immunostaining. The final diagnosis was HTT of the thyroid gland. Next-generation sequencing genetic analysis of a surgical specimen revealed no pathologic mutations, including BRAF, H/K/NRAS, or RET-PTC fusions. The PAX8-GLIS3 fusion was detected by RT-PCR.ConclusionsA rare case of HTT was demonstrated through imaging, cytologic, histologic and molecular investigations. PAX8-GLIS3 fusion detected by RT-PCR and Sanger sequencing was confirmed to be a genetic hallmark of HTT.