FGFR1 Research Articles

Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions: reevaluation of the defining characteristics in a registry-based cohort

In a registry-based analysis of 135 patients with “myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions” (MLN-TK; FIP1L1::PDGFRA, n = 78; PDGFRB, diverse fusions, n = 26; FGFR1, diverse, n = 9; JAK2, diverse, n = 11; ETV6::ABL1, n = 11), we sought to evaluate the disease-defining characteristics. In 81/135 (60%) evaluable patients, hypereosinophilia (>1.5 × 109/l) was observed in 40/44 (91%) FIP1L1::PDGFRA and 7/7 (100%) ETV6::ABL1 positive patients but only in 13/30 (43%) patients with PDGFRB, FGFR1, and JAK2 fusion genes while 9/30 (30%) patients had no eosinophilia. Monocytosis >1 × 109/l was identified in 27/81 (33%) patients, most frequently in association with hypereosinophilia (23/27, 85%). Overall, a blast phase (BP) was diagnosed in 38/135 (28%) patients (myeloid, 61%; lymphoid, 39%), which was at extramedullary sites in 18 (47%) patients. The comparison between patients with PDGFRA/PDGFRB vs. FGFR1, JAK2, and ETV6::ABL1 fusion genes revealed a similar occurrence of primary BP (17/104, 16% vs. 8/31 26%, p = 0.32), a lower frequency (5/87, 6% vs. 8/23, 35%, p = 0.003) of and a later progression (median 87 vs. 19 months, p = 0.053) into secondary BP, and a better overall survival from diagnosis of BP (17.1 vs. 1.7 years, p < 0.0008). We conclude that hypereosinophilia with or without monocytosis and various phenotypes of BP occur at variable frequencies in MLN-TK.

Explainable artificial intelligence to predict and identify prostate cancer tissue by gene expression

Background and Objective: Prostate cancer is one of the most prevalent forms of cancer in men worldwide. Traditional screening strategies such as serum PSA levels, which are not necessarily cancer-specific, or digital rectal exams, which are often inconclusive, are still the screening methods used for the disease. Some studies have focused on identifying biomarkers of the disease but none have been reported for diagnosis in routine clinical practice and few studies have provided tools to assist the pathologist in the decision-making process when analyzing prostate tissue. Therefore, a classifier is proposed to predict the occurrence of PCa that provides physicians with accurate predictions and understandable explanations.Methods: A selection of 47 genes was made based on differential expression between PCa and normal tissue, GO gene ontology as well as the literature to be used as input predictors for different machine learning methods based on eXplainable Artificial Intelligence. These methods were trained using different class-balancing strategies to build accurate classifiers using gene expression data from 550 samples from ’The Cancer Genome Atlas’. Our model was validated in four external cohorts with different ancestries, totaling 463 samples. In addition, a set of SHapley Additive exPlanations was provided to help clinicians understand the underlying reasons for each decision.Results: An in-depth analysis showed that the Random Forest algorithm combined with majority class downsampling was the best performing approach with robust statistical significance. Our method achieved an average sensitivity and specificity of 0.90 and 0.8 with an AUC of 0.84 across all databases. The relevance of DLX1, MYL9 and FGFR genes for PCa screening was demonstrated in addition to the important role of novel genes such as CAV2 and MYLK.Conclusions: This model has shown good performance in 4 independent external cohorts of different ancestries and the explanations provided are consistent with each other and with the literature, opening a horizon for its application in clinical practice. In the near future, these genes, in combination with our model, could be applied to liquid biopsy to improve PCa screening.

Screening the Chemical Composition and Bioactivity of Cerasus serrulata Flower Extracts Using Two Extraction Methods

Background Cerasus serrulata has an excellent antioxidative, antibacterial, and anti-alopecia effect, but its active ingredients and the underlying mechanism are currently unclear, in addition to the fact that the active products vary with the extraction methods. Aim Therefore, this study aims to clarify the active components and antioxidative property of C. serrulata extracted by two different methods (low-temperature vacuum extraction and ethanol extraction) and explore its potential biofunction. Materials and Methods First, the compositions in extracts were characterized by gas chromatographic mass spectrometry (GC-MS) and liquid chromatography-mass spectrography (LC-MS). Then, the antioxidative activity of both extracts was evaluated using free radical scavenging assay (ABTS) and ORAC. Next, the antibacterial activity of both extracts was measured via a bacteriostatic circle experiment. The anti-alopecia effects of both extracts were studied using a mouse model of alopecia. The hematoxylin and eosin (H&amp;E) staining was used to evaluate hair follicles; vascular endothelial-derived growth factor (VEGF) and interleukin-6 (IL-6) expression in mouse skin were evaluated by immunohistochemistry, while β-catenin expression in tissues was used by immunofluorescence. The expression of FGFR and insulin-like growth factor 1 (IGF-1) in the skin was determined to evaluate using real-time quantitative polymerase chain reaction (RT-qPCR). The levels of DHT and 5α-reductase in the experimental mice were determined by ELISA. Results The GC-MS analysis results showed that the extracts were rich in alkanes and aromatic compounds, specifically benzaldehyde, which was the highest component in the cold vacuum extraction, while pipecolic acid was the main component in the ethanol extract. This indicates that C. serrulata has components that exhibit strong inhibitory ability against the growth of five common bacterial strains, expressing excellent free radical scavenging ability. In addition, the active ingredients in C. serrulata were found to significantly deregulate the protein levels in the skin of androgenetic alopecia (AGA) mice, with the levels of IL-6. They were also able to significantly inhibit VEGF reduction. The β-catenin was being significantly upregulated in skin tissue. In addition, FGFR and IGF-1 gene expression also increased. The 5α-reductase and dihydrotestosterone (DHT) can be reduced in the C. serrulata treatment. Conclusion Our data indicate that C. serrulata extract could be beneficial supplements of natural antioxidative, antibacterial, and anti-alopecia agents.

KLF2 regulates stemness of human mesenchymal stem cells by targeting FGFR3

ABSTRACT Mesenchymal stem cells (MSCs) are an attractive source of pluripotent cells for regenerative therapy; however, maintaining stemness and self-renewal of MSCs during expansion ex vivo is challenging. For future clinical applications, it is essential to define the roles and signaling pathways that regulate the fate of MSCs. Based on our earlier finding that Krüppel-like factor 2 (KLF2) participates in maintaining stemness in MSCs, we examined further the role of this factor in intrinsic signaling pathways. Using a chromatin immunoprecipitation (ChIP)-sequence assay, we found that the FGFR3 gene is a KLF2 binding site. Knockdown of FGFR3 significantly decreased the levels of key pluripotency factors, enhanced the expression of differentiation-related genes and down-regulated colony formation of human bone marrow MSCs (hBMSCs). Using alizarin red S and oil red O staining, we found that knockdown of FGFR3 inhibited the osteogenic and adipogenic ability of MSCs under conditions of differentiation. The ChIP-qPCR assay confirmed that KLF2 interacts with the promoter regions of FGFR3. Our findings suggest that KLF2 promotes hBMSC stemness by direct regulation of FGFR. Our findings may contribute to enhanced MSC stemness by genetic modification of stemness-related genes.

RNA Sequencing Reveals Novel Oncogenic Fusions and Depicts Detailed Fusion Transcripts of FN1-FGFR1 in Phosphaturic Mesenchymal Tumors

Phosphaturic mesenchymal tumors (PMTs) are rare neoplasms of soft tissue or bone. Although previous studies revealed that approximately 50% of PMTs harbor FN1::FGFR1 fusions, the molecular mechanisms in the remaining cases are largely unknown. In this study, fusion genes were investigated using RNA-based next-generation sequencing in 76 retrospectively collected PMTs. Novel fusions were validated with Sanger sequencing and fluorescence in situ hybridization. Fusion genes were detected in 52/76 (68.4%) PMTs, and 43/76 (56.6%) harbored FN1::FGFR1 fusions. Fusion transcripts and breakpoints of the FN1::FGFR1 fusions were diverse. The most common fusion transcript was between exon 20 of FN1 and exon 9 of FGFR1 (7/43, 16.3%). The most upstream breakpoint of the FN1 gene was located at the 3′ end of exon 12, and the most downstream breakpoint of the FGFR1 gene was at the 5′ end of exon 9, suggesting the inessential nature of the third fibronectin-type domain of FN1 and the necessity of the transmembrane domain of FGFR1 in the FN1::FGFR1 fusion protein, respectively. Moreover, the reciprocal FGFR1::FN1 fusions, which had not been identified in previous studies, were detected in 18.6% (8/43) of FN1::FGFR1 fusion-positive PMTs. Novel fusions were identified in 6/76 (7.9%) FN1::FGFR1 fusion-negative PMTs, including 2 involving FGFR: FGFR1::USP33 (1/76, 1.3%) and FGFR1::TLN1 (1/76, 1.3%). Other novel fusions identified were the PDGFRA::USP35 (1/76, 1.3%), SPTBN1::YWHAQ (1/76, 1.3%), GTF2I::RALGPS1 (1/76, 1.3%), and LTBP1::VWA8 (1/76, 1.3%) fusions. In addition to these novel fusions, FN1::FGFR2 (1/76, 1.3%), NIPBL::BEND2 (1/76, 1.3%), and KIAA1549::BRAF fusions (1/76, 1.3%) were also identified in FN1::FGFR1-negative cases arising from the thigh, ilium, and acetabulum, respectively. The frequency of oncogenic fusions was significantly higher (P = .012) in tumors derived from extremities (29/35, 82.9%) compared with other locations (23/41, 56.1%). No significant correlation was identified between fusions and recurrence (P = .786). In conclusion, we report fusion transcripts and breakpoints of FN1::FGFR1 in PMTs in detail, providing insights into fusion protein functions. We also revealed that a considerable proportion of PMTs without FN1::FGFR1 fusion carried novel fusions, providing further insight into the genetic basis of PMTs.

The Significance of Human Papillomavirus Receptors Related Genetic Variants in Cervical Cancer Screening.

To investigate the relationship between single nucleotide polymorphisms (SNPs) in human papillomavirus (HPV) receptor associated genes and HPV susceptibility and clinical outcomes in Chinese women, from October 2016 to March 2020, a total of 3,066 women were recruited for a 3-year prospective population-based cervical cancer screening clinical trial. The primary endpoint was histological cervical intraepithelial neoplasia 2 and worse (CIN2+). Twenty-nine SNPs of HPV receptor associated genes on women with available cytology residual samples at baseline were detected using MALDI-TOF MS. Eligible data were available for 2,938 women. Rs16894821 (GG versus AA, OR =1.71 [1.08 to 2.69]) and rs724236 (TT versus AA, OR = 1.73 [1.14 to 2.62]) in SDC2 were significantly related to the HPV susceptibility. And rs2575712 (TT versus GG, OR = 2.78 [1.22 to 6.36]) in SDC2 was associated with increased HPV 16/18 susceptibility. Four SNPs (rs1047057 and rs10510097 in FGFR2 gene, rs2575735 in SDC2 gene, and rs878949 in HSPG2 gene) were significantly associated with persistent HPV infection. Importantly, the genotypes of rs16894821 under recessive model (GG versus AA/AG, OR = 2.40 [1.12 to 5.15]) in SDC2 and rs11199993 under dominant model (GC/CC versus GG, OR = 1.64 [1.01 to 2.68]) in FGFR2 were significantly associated with the disease progression. Finally, SNPs showed comparable efficacy in detecting CIN2+ for the women infected with non-HPV16/18 compared with cervical cytology (sensitivity: 0.51 [0.36 to 0.66] versus 0.44 [0.30 to 0.60], specificity: 0.96 [0.96 to 0.97] versus 0.98 [0.97 to 0.99], positive predictive value: 0.23 [0.15 to 0.33] versus 0.33 [0.22 to 0.47], and negative predictive value: 0.99 [0.98 to 0.99] versus 0.99 [0.98 to 0.99]). SNPs in HPV receptor related genes may influence HPV susceptibilities and clinical outcomes in Chinese women. IMPORTANCE Virus receptors are known to mediate virus attachment and further lead to virus infection of the host cells. In the current study, we investigated the relationship between single nucleotide polymorphisms (SNPs) in human papillomavirus (HPV) receptor associated genes and HPV susceptibility and clinical outcomes in Chinese women, and to explore the new triaging strategy for non-16/18 high-risk HPV infection.

Hypochondroplasia gain-of-function mutation in FGFR3 causes defective bone mineralization in mice.

Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.

Futibatinib (Lytgobi®)

Futibatinib (Lytgobi®)

The management of achondroplasia in Italy: results from a Delphi panel based on real-world experience.

Achondroplasia is a rare genetic disorder caused by a mutation in the FGFR3 gene, leading to skeletal changes and other systemic complications that greatly impact the patient's quality of life. There currently are differences in achondroplasia patients' management among countries and centers within the same country. A group of Italian experts discussed the best practice and the current unmet needs in the management of patients with achondroplasia though a two-round Delphi panel, between September and November 2022. The Delphi survey consisted of 32 questions covering organizational aspects, diagnosis and follow-up, and management of achondroplasia patient, and was shared among 54 experts from 25 different centers in Italy. The consensus was determined on the basis of the percentage of agreement or disagreement to each statement on a 5-point Likert scale. Pediatricians (including specialists in pediatrics, medical genetics, and pediatric endocrinology) orthopedics and medical geneticists were the most represented specialists accounting for 64%, 9% and 9% of participants, respectively. The panel highlighted the need for standardized procedures to identify reference centers, the crucial role of multidisciplinary team, and effective communication among centers (Hub and Spoke model) as the essential organizational features; the importance of genetic counseling, presence of a psychologist, and clear communication during prenatal diagnosis as main points for diagnosis; early intervention by different specialists, personalized care, and promotion of a healthy lifestyle as major points for patient management. To ensure an adequate continuity of care over the whole lifespan of a patient with achondroplasia a shared model for patient management is suggested by Italian specialists.

Frequency of PDGFRA, PDGFRB and FGFR1 Gene Rearrangements in Patients with Eosinophilia and Their Clinico-Haematologic Parameters

Objective: To determine the frequency and clinico-haematological features of PDGFRA, PDGFRB and FGFR1 gene rearrangements in patients with persistent Eosinophilia using Fluorescence in situ hybridization.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Dec 2018 to Dec 2019.&#x0D; Methodology: All Patients who presented to AFIP having absolute eosinophil count &gt;1.5x109/L persistent for over six months or with Myeloid or Lymphoid neoplasms with persistent Eosinophilia were studied. Patients having reactive Eosinophilia and those on treatment were excluded. Interphase FISH studies were performed. In addition, 2.5ml of sodium heparin blood was taken. After the denaturation of DNA, slides were set up according to standard protocol. FIP1L1/CHIC2/PDGFRA dual colour probe was applied for PDGFRA, 5q32 PDGFRA break apart probe for PDGFRB and XL FGFR1 break apart probe for FGFR1 gene rearrangement.&#x0D; Results: A total of 60 patients were included in the study. Of these, 50(83.3%) were males, and 10(16.7%) were females, with an average absolute Eosinophilia count of 5.92±7.10x109/L. The only rearrangement detected in patients with Eosinophilia was FIPILI-PDGFRA gene fusion, detected in 20% of the patients. No other rearrangement was found.&#x0D; Conclusion: PDGFRA, PDGFRB and FGFR1 mutations are rare yet most prominent in patients with clonal Eosinophilia. About 80% of eosinophilic patients were found to have idiopathic Eosinophilia, which requires further consideration to address the disease prevalence.

Genomically matched therapy in refractory colorectal cancer according to ESMO Scale for Clinical Actionability of Molecular Targets: experience of a comprehensive cancer center network.

Efficiency of expanded genomic profiling (EGP) programmes in terms of final inclusion of patients in genomically matched therapies is still unknown. Fit patients with advanced and refractory colorectal cancer (CRC) were selected for an EGP programme. Next-generation sequencing (NGS) analysis from formalin-fixed paraffin-embedded tumour samples was performed. The purpose was to describe the prevalence of genomic alterations defined by the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT), as well as the percentage of patients finally included in genomically guided clinical trials. In total, 187 patients were recruited. Mutational profile was obtained in 177 patients (10 patients were failure due to insufficient tumour sample), copy number alterations in 41 patients and fusions in 31 patients. ESCAT-defined alterations were detected in 28.8% of the intention-to-analyse population. BRAF V600E was clustered in ESCAT I, with a prevalence of 3.7%, KRAS G12C and ERBB2 amplification were clustered in ESCAT II, whose prevalence was 4.2% and 1.6%, respectively. Most alterations were classified in ESCAT III (mutations in ERBB2, PIK3CA or FGFR genes and MET amplification) and IV (mutations in BRAF non-V600E, ERBB3, FBXW7, NOTCH, RNF43), with a single prevalence under 5%, except for PIK3CA mutation (9%).The final rate of inclusion into genomically guided clinical trials was 2.7%, including therapies targeting BRAF V600E or RNF43 mutations in two patients each, and ERBB2 mutation in one patient. In conclusion, EGP programmes in patients with advanced CRC are feasible and identify a subset of patients with potentially druggable genomic alterations. However, further efforts must be made to increase the rate of patients treated with genomically guided therapies.

FGFR1 gene fusions in a subset of pediatric mesenchymal tumors: Expanding the genetic spectrum of tumors sharing histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms.

As the classification of kinase-driven spindle cell tumors continues to evolve, we describe the first series of pediatric mesenchymal tumors harboring FGFR1 gene fusions that share histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms. Herein, we present three cases of FGFR1-rearranged pediatric mesenchymal tumors, including one case with FGFR1::PARD6B gene fusion and two cases with FGFR1::EBF2 gene fusion. The tumors involved infants ranging from 3 to 9 months in age with a male-to-female ratio of 2:1. All tumors involved the deep soft tissue of the gluteal, pelvic, or perirectal region. Histologically, the tumors comprised a cellular spindle cell neoplasm with primitive stellate cells, focal myxoid stroma, focal epithelioid features, no necrosis, and occasional mitotic figures (2-6 per 10 high-power field). By immunohistochemistry, the neoplastic cells focally expressed CD34 but lacked expression of S100 protein, SMA, desmin, myogenin, MyoD1, pan-TRK, and ALK. These three cases, including a case with long-term clinical follow-up, demonstrate that FGFR1 fusions occur in a subset of newly described pediatric kinase-driven mesenchymal tumors with locally aggressive behavior. Importantly, knowledge of these genetic alterations in this spectrum of pediatric tumors is key for diagnostic and targeted therapeutic purposes.

Landscape of FGFR2/3 alterations in hepatobiliary malignancies.

e16152 Background: Genomic aberrations (GAs) in FGFRs are involved in the pathogenesis of Hepatobiliary Malignancies, and clinical trials have shown efficacy of FGFR inhibitors in treating hepatobiliary malignancies patients with FGFR GAs. However, the molecular profile of FGFR2/3 in hepatobiliary malignancies is still lacking. Here, we focused on mutational landscape of FGFR2/3 GAs in hepatobiliary malignancies. Methods: We prospectively sequenced 876 tumors and matched blood DNA from Chinese and TCGA patients with hepatobiliary malignancies using next-generation sequencing techniques. Comprehensive molecular characterization was analyzed. Results: 13.2% (116/876) hepatobiliary malignancies carried FGFR2/3 GAs, including somatic mutation (56, 6.4%), CNV (41, 4.7%), and fusion (23, 2.62%). In FGFR2/3 mutation cohorts, the five most frequently somatic mutated genes were TP53 (46%), NCOR2 (26%), LRP1B (18%), KMT2C (17%), and FAT1 (16%), except for FGFR2 and FGFR3 gene. For FGFR2/3 wildtype cohorts, the five most frequently mutated genes were TP53 (46%), NCOR2 (13%), CTNNB1 (12%), LRP1B (12%), and ARID1A (10%). 77 mutated genes were significant difference between FGFR2/3 mutation and FGFR2/3 wildtype cohorts, such as NCOR2, KMT2C, FAT1, ROS1 and KMT2B, excluding the FGFR2 and FGFR3 gene. In addition, patients with FGFR2/3 GAs harbored a significantly higher TMB than those in patients without FGFR2/3 GAs (12.6 vs 9.0, p=0.003). There was no significant difference in genomic instability between the FGFR3 mutant group and the wild group (p&gt;0.05), including ploidy, wGII, and ITH. Conclusions: FGFR2/3 GAs are frequently altered in hepatobiliary malignancies. Comprehensive genomic profiling of FGFR2/3 GAs highlights the clinical and therapeutic implication. There were characterized the genomic differences and similarities, stratified by the FGFR2/3 status, which may reflect the hepatobiliary malignancies patients with FGFR2/3 GAs harbored specific molecular mechanism.

CtDNA-based genomic landscape analysis to evaluate molecular brake and gatekeeper mutations in FGFR2 alterations.

3049 Background: FGFR2 and FGFR3 (FGFR2/3) gene alterations are oncogenic drivers found across various cancer types and have been successful targets of drug development. Several FGFR inhibitors (FGFRi) are approved for clinical use, and most are ATP-competitive, reversible inhibitors (1st-gen). Efficacy of the 1st-gen FGFRi can be limited by acquired resistance mutations in the FGFR kinase domain, typically molecular brake or gatekeeper mutations, which disrupt compounds binding through conformational changes or through direct steric hindrance, respectively. Irreversible (covalent) FGFRi, such as futibatinib, are in development to overcome kinase domain resistance mutations. However, preclinical evidence indicates that some of the known resistance mutations (eg. V564L) remain insensitive to futibatinib. Methods: To assess the landscape of FGFR-driven cancers and to better understand the prevalence of molecular brake and gatekeeper mutations, we analyzed the distribution of FGFR2/3 alterations in a large clinical genomic research database. This ctDNA-based genomic database was selected for the analysis given that liquid biopsy is a fit-for-purpose method to detect acquired resistance mutations. Results: We identified over 2,100 patients with FGFR2/3 alterations. Non-small cell lung cancer (NSCLC), breast, bladder, and cholangiocarcinoma (CCA) had the highest number of patients with FGFR2/3 alterations. 36% of all FGFR2 alterations across all cancer indications consisted of molecular brake or gatekeeper mutations. This finding was most enriched in CCA and breast cancer where 51% and 57%, respectively, of FGFR2 alterations were molecular brake or gatekeeper mutations. In breast cancer patients, molecular brake mutations were common and represent activating mutations in FGFRi-naïve patients. A small number of FGFR3 gene fusions were detected in CCA patients who had significantly shorter time on 1st-line treatment and trended toward shorter overall survival when compared to patients with FGFR2 fusions. We also identified CCA patients with FGFR2 fusions and one or more concurrent FGFR2 SNVs; primarily those in the kinase domain and associated with FGFRi resistance. Most of these putative cases of acquired resistance were polyclonal, with 2 or more (up to 8) FGFR2 SNVs coexisting with the fusion. Bladder cancer and NSCLC had the highest numbers of FGFR3 alterations. FGFR2/3 alterations were typically found without other driver mutations in CCA, bladder, and NSCLC patients. However, in breast there was a large overlap between FGFR2/3 alterations and other drivers. Conclusions: Herein, we identify a cohort of patients with FGFR2/3 molecular brake and gatekeeper mutations that may benefit from next-generation FGFR2/3-targeted therapies designed to inhibit a broad spectrum of both primary and secondary resistance mutations.

P-137 Phase 1/1b study evaluating KIN-3248, a next-generation, irreversible pan-FGFR inhibitor (FGFRi), in patients (pts) with advanced cholangiocarcinoma (CCA) and other solid tumors harboring FGFR2 and/or FGFR3 gene alterations

P-137 Phase 1/1b study evaluating KIN-3248, a next-generation, irreversible pan-FGFR inhibitor (FGFRi), in patients (pts) with advanced cholangiocarcinoma (CCA) and other solid tumors harboring FGFR2 and/or FGFR3 gene alterations

Investigating alterations in cancer driver genes and other potentially targetable mutations in patients with intrahepatic cholangiocarcinoma (iCCA) treated on the randomised phase III multicentre BILCAP clinical trial.

4019 Background: The BILCAP clinical trial established adjuvant capecitabine as the standard of care treatment in patients with resected biliary tract cancer. Translational work to investigate the role of cancer driver genes and other potentially targetable mutations in patients enrolled on BILCAP was performed, with this analysis focusing on patients with intrahepatic cholangiocarcinoma (iCCA). Methods: Archived fixed formalin (FFPE) tissue samples were collected from consented BILCAP patients. These samples underwent DNA and RNA extraction followed by low-pass whole genome sequencing (lp-WGS), targeted gene sequencing (TGS) and RNA sequencing (RNAseq) for copy number (CN) analysis, mutation analysis and gene fusion analysis. Results: 84 of the 447 BILCAP patients had iCCA; 45 successfully underwent lp-WGS and RNAseq, of whom 36 also underwent TGS. The median age was 61 years (95% CI 57.8 – 64.2), 55.6% were female and 23 received capecitabine (51.1%). FGFR2 gene fusions were present in 9 patients (20.0%), as were fusions in NTRK1 (n = 3, 6.7%), FGFR1 (n = 3, 6.7%), FGFR3 (n = 2, 4.4%) and FGFR4 (n = 2, 4.4%). Commonly mutated driver genes included ROS1 (n = 12, 33.3%), MET (n = 10, 27.8%) and ALK (n = 7, 19.4%) with known pathogenic variants seen in IDH1 (n = 4, 11.1%; total number of mutations = 7, 19.4%), BRAF (n = 2, 5.6%; total n = 6, 16.7%), FGFR2 (n = 1, 2.7%; total n = 8, 22.2%), FGFR3 (n = 1, 2.7%, total n = 6, 16.7%) , IDH2 (n = 1, 2.7%; total n = 6, 16.7%) and EGFR (n = 1, 2.7%; total n = 4, 11.1%). Commonly amplified (CN ≥ 4, ploidy &lt; 3) genes included NTRK1 (n = 9, 20.0%), ERBB2 (n = 8, 17.8%), and MET (n = 3, 6.7%). Most of the alterations investigated in this cohort did not significantly affect recurrence risk or overall survival (OS), including FGFR2 fusions (OS HR 1.23, p = 0.695; recurrence HR 1.32 p = 0.555). However, the presence of a FGFR3 fusion gene significantly reduced OS (OS HR 6.57, p = 0.0091; recurrence HR 3.71, p = 0.0734), and having ≥ 4 copies of either NTRK1 (OS HR 3.55, p = 0.0027; recurrence HR 3.48, p = 0.0019) or MET (OS HR 6.06, p &lt; 0.001; recurrence HR 6.05 p &lt; 0.001) significantly reduced OS and increased the risk of recurrence. Conclusions: The BILCAP cohort shows a wide variety of driver and potentially targetable mutations in unselected iCCA patients, comparable to previous early-stage biliary tract cancer datasets. Of note, patients with FGFR3 fusions, MET amplification or NTRK1 amplification had significantly shorter OS, and patients with MET or NTRK1 amplification had significantly reduced OS and significantly increased risk of disease recurrence. MET amplification, NTRK1 amplification and FGFR3 fusions may be important indicators in determining prognosis, and could provide attractive targets for future targeted anti-cancer therapy in iCCA. Clinical trial information: EUCTR2005-003318-13.

AI-based identification of FGFR3 mutation status from routine histology slides of muscle-invasive bladder cancer.

e16580 Background: Gain of function mutations of the FGFR3 gene have been reported to be important driver mutations in a subset of muscle-invasive bladder urothelial cancer (MIBC). Subsequently, gene specific kinase inhibitors (e.g., erdafitinib) have been developed to target FGFR3 mutant metastasized MIBC with promising clinical activity. FGFR3 mutational status testing is required for erdafitinib treatment, but FGFR3 mutations are rare in metastatic MIBC (10-15%). Thus, cheap and fast pre-screening tools such as artificial intelligence (AI) based image classifiers could help to reduce turnover times of FGFR3 mutational testing and save costs arising from universal mutational testing. Herein, we propose an AI-based approach to identify MIBC FGFR3 mutated patients on routine histological slides. Methods: Slides from 612 patients with MIBC from two different sources (CCC-EMN Erlangen cohort n = 239, mutant = 20; TCGA-BLCA n = 373, mutant = 50) were used. The CCC-EMN cohort was used as a discovery set while TCGA-BLCA was used as an independent validation set. A two-step AI model was used to 1) extract features from each slide and 2) predict FGFR3 status from these features. The AUC, sensitivity and specificity were used as metrics to assess the performance of our model. Most predictive regions of FGFR3 mutation were reviewed by an expert pathologist. Results: The model reached an AUC/Sensitivity/Specificity of 0.82 [0.76-0.87] / 0.90 [0.82-0.97] / 0.57 [0.53-0.62] on TCGA-BLCA, substantially improving upon results previously reported on the same cohort (Loeffler et al. 2021: AUC = 0.70). Most predictive regions of FGFR3 mutation revealed monomorphous conventional urothelial tumor morphology and low desmoplastic tumor stroma content, while least predictive regions contained pleomorphic tumor morphology and high amounts of desmoplastic tumor content. Conclusions: By ruling out 57% of MIBC FGFR3 wild type tumors with high sensitivity, our model is the first to reach valuable performance for clinical use. Consequently, this study paves the way for the development of FGFR3 pre-screening tools to ease the management of patients with MIBC.

Accuracy of genome-wide mutational analysis for tumor-informed ctDNA-guided MRD monitoring in bladder cancer.

4589 Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard management of localized muscle-invasive bladder cancer (MIBC). However, about 45% of patients develop metastases within 2 years after RC. Biomarkers for treatment efficacy evaluation and early detection of minimal residual disease (MRD) are needed for earlier treatment initiation and monitoring of treatment response. Tumor-informed detection of mutations in cell-free DNA (cfDNA) has shown promising results to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied a whole-genome sequencing (WGS) approach to monitor circulating tumor DNA (ctDNA) for sensitive ctDNA detection. Methods: 110 MIBC patients undergoing NAC and RC were enrolled. cfDNA was extracted from ~1mL plasma (n=978) and procured from longitudinal plasma sampling during NAC and pre-RC and post-RC. WGS of tumor/germline pairs (30x/20x) and plasma cfDNA (&gt;20x) was performed, facilitating detection of genome-wide genomic alterations and quantification of ctDNA using the C2inform method. Results: For each patient we developed a tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were used for detection and measuring ctDNA levels in low-input blood samples by WGS. Post-RC ctDNA analysis identified patients with recurrence with 78% sensitivity and 95% specificity and with a median lead time over radiographic imaging of 118 days (full follow-up included). When restricting follow-up time to 12 months following the latest ctDNA test, a sensitivity of 86% and specificity of 94% was achieved. ctDNA status was associated with recurrence-free (p&lt;0.0001) and overall survival (p&lt;0.0001). Furthermore, ctDNA clearance during NAC was also associated with recurrence-free survival (p=0.0051). The APOBEC associated signatures SBS2 and SBS13 were identified as the primary contributors to the mutational landscape of the primary tumors and TP53, KMT2D, PIK3CA, RB1 and KDM6A were the main affected driver genes. Half of the tumors displayed whole genome doubling. Moreover, our analysis of plasma samples after treatment showed the presence of chemotherapy-induced mutational signatures not present in the primary tumor, along with increases in copy numbers on chromosomes 19q and 20, and a focal amplification of the FGFR3 gene on chromosome 4. Conclusions: Our results highlight the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring.

A Review on Different Manifestations of Crouzon Syndrome

Introduction: Crouzon syndrome (CS), the most common craniosynostosis condition, which could lead to several developmental complications. This study aimed to review the different manifestations of CS. Material and Methods: In order to find the relevant articles, the databases of PubMed, Scopus, Web of Science, and Cochrane Library were searched using the term “Craniofacial Dysostosis” and its relevant entry terms. All English-language articles regarding the CS were included in the study. After removing the duplicate articles, two authors independently screened the title and abstracts of the included articles. Disagreements were resolved through voting and discussion with the third author. Then full-text of articles were screened and the articles were categorized depending on regarding their main topic. Results: The search yielded 449 results in different databases. After removing the duplicates, 331 results remained. Then, 182 were excluded as not completely relevant by screening the abstracts. The remaining 149 studies were assessed for the eligibility criteria. Of them, 74 were excluded due to the following reasons: (1) unavailable full text; (2) discussing other types of craniosynostoses syndromes; and (3) not having clear results. Finally, 75 studies which were included in this study. Conclusion: CS is caused by mutations in the FGFR2 gene and is inherited in an autosomal dominant pattern. Diagnosis is based on the characteristic physical features, as well as imaging studies and genetic testing. Treatment involves surgery to correct the craniosynostosis and facial abnormalities. Early and appropriate treatment can help to improve the quality of life for affected individuals.

179P Developing the AI program for TNBC subtyping

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis and can be classified into different molecular subtypes. There is a need to develop a computer-aided diagnosis system with artificial intelligence (AI) techniques to increase the accuracy of evaluating suspicious breast lesions and identifying subtypes. A multi-gene analysis panel consisting of 14 genes and 2 reference genes was designed from the TCGA, and cBioportal datasets, which were determined in accordance with in silico analyses in 488 TNBC patients (TCGA:123, cBioportal:365). In the AI part of the study, a convolutional neural network model with 1,838,915 parameters was trained to classify Ultrasound (US) images as “normal, benign, malignant.” In the model, the images of the patients were trained with 1000 epochs. Expression levels of related genes in paraffin-embedded tumors and normal tissues of 38 patients with TNBC were investigated by the RT-PCR method. When the gene expression differences of the tumor and normal tissues of the patients were compared, AR (p=0.013), ER (p=0.025), PGR (p=0.007), FOXA1 (p=0.0154), CXCL11 (p=0.0037), IDO1 (p=0.0005), ADH1B (p=0.018), ADIPOQ (p<0.0001) and CHAD (p=0.0017) genes were found to have a statistically significant difference in expression compared to normal tissue. While no significant expression difference was detected in FOXC1 (p=0.3914), CXCL13 (p=0.2138), LAMP3 (p=0.2576), FGFR2 (p=0.077) and DHRS2 (p=0.715) genes. As a result, it was found that 68.5% of the patients showed a single subtype-specific expression profile (10 BLIA, 3 BLIS, 1 MES, and 12 LAR subtypes), and 31.5% of the patients showed an expression profile of more than one subtype. A random forest model was chosen among the trained models, and in the first stage, 81% accuracy was obtained in the subtyping of TNBC and 83% in the classification of US images. It sheds light on the heterogeneous structure and the role of molecular subtyping in the tumorigenesis of TNBC patients and may contribute to routine clinical practice and the regulation of targeted therapy protocols.