FGFR3 Mutations Research Articles

BIOM-49. FGFR ALTERATIONS IN GLIOMAS; CLINICAL, RADIOGRAPHIC AND MOLECULAR CHARACTERIZATION

Abstract BACKGROUND FGFR-TACC is the most prevalent type of gene fusion in adult gliomas. It is present in 3-6% of IDH WT (wild-type) gliomas. Due to their histopathological and molecular features, it has been suggested that FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. F3T3 are potent oncogenes that confer sensitivity to FGFR inhibitors. Preliminary data showed promising results of these alterations as actionable targets by FGFR inhibitors. METHODS We retrospectively analyzed molecular data on 912 glioma patients treated at our institution from 2014 to 2023. RESULTS We identified 22 gliomas (2.5%) with FGFR alterations. Median age was 60 years, 14 were female. 14 patients (64%) self-identified as Hispanic. 18 had a diagnosis of glioblastoma, 3 had other gliomas, and one had a low-grade neuroepithelial neoplasm. Ten patients had tumor location in the temporal lobe, 6 frontal, 5 parietal, 3 occipital, 1 multifocal, and 1 basal ganglia. 19 patients had enhancing tumors, while three were non-enhancing. The most common FGFR alteration was FGFR3-TACC3 fusion in 14/22 patients, 6 had FGFR mutations (either single base or amino acid substitutions), one had FGFR-LMNB2 fusion, and one had FGFR3 amplification. 86.4% of our patient cohort (19/22) had a confirmed TERT promoter mutation, while 100% of GBM patients with known TERT promoter status had an associated TERT promoter mutation. 10/22 had CDKN2A/B alterations. One patient had pathology-proven systemic metastases. CONCLUSIONS FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. In our cohort, 100% of the patients with F3T3-positive glioblastomas also had an associated TERT promoter mutation. To our knowledge, this is one of the larger cohorts showing the association between FGFR3 and TERT promoter alterations in glioblastomas. RESULTS will be validated through the upcoming clinical trial ETCTN 10559, including tumor evolution and mechanisms of resistance.

Clinical significance of molecular subgroups of polymorphous low-grade neuroepithelial tumor of the young (PLNTY): A small single institutional case series and integrated analysis

IntroductionPolymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described entity. The clinicopathological features and prognosis of the molecular subgroups of these rare tumors is poorly understood. In this study, we presented a small case series of three new cases and integrated the data with published cases in the literature to characterize the similarities and differences of molecular subgroups of PLNTY. MethodsWe searched our institutional archive for PLNTY cases and searched PubMed and Web of Science for relevant data. Demographic, clinical, radiologic, histopathological, molecular, and follow-up data of our four cases with published cases were integrated for final analyses. ResultsWe identified three institutional cases of PLNTY. The median age of our patients was 17 years (range: 13–42). All patients had a prior history of chronic seizures and all had tumors affecting the temporal lobes. Histopathologically, all cases showed oligodendroglial-like morphology with intratumoral calcifications and at least partially infiltrative growth patterns. Tumor cells were immunoreactive with CD34 and GFAP. Genetically, all cases harbored BRAF V600E mutations. Integrated analyses, including a total of 67 cases, demonstrated that PLNTYs with FGFR2 mutation were significantly younger (median age 11.0 years) than those with BRAF V600E or FGFR3 fusions (median age 41.0 and 16.0 years, respectively). All BRAF V600E-positive PLNTYs were free of tumor recurrence, while four of PLNTYs in other molecular subgroups developed tumor recurrence by imaging. ConclusionOur study suggests that PLNTYs have distinct clinicopathological features and are driven by genetic alterations in the MAPK pathway. The molecular subgroups of PLNTYs share similar findings, but also demonstrate distinct patient demographics.

Ipsilateral synchronous papillary renal neoplasm with reverse polarity and urothelial carcinoma in a renal transplant recipient: a rare case report with molecular analysis and literature review

BackgroundRenal transplant recipients (RTRs) have a 3- to 5-fold higher risk of developing malignant tumors than the general population, with new malignant tumors after transplantation considered to be the leading cause of death in RTRs. In pathological practice, it is rare for neoplasms with different histology to be located in the same organ. We report the first case of a synchronous papillary renal neoplasm with reverse polarity (PRNRP) and urothelial carcinoma (UC) in the ipsilateral kidney in an RTR. Molecular detection was conducted by next-generation sequencing.Case presentationA 68-year-old female suffered from uremia 19 years ago and underwent renal transplantation (RT) after receiving dialysis for 6 months. Hematuria occurred one month ago and an enhanced CT showed that there were two abnormal density foci in the middle and lower parts of the autologous left kidney. A laparoscopic left nephrectomy and ureterectomy were performed. Gross examination revealed a mass (I) in the left renal parenchyma, 2*1.8*1.5 cm in size, that protruded from the renal capsule, and a cauliflower-like mass (II), 5*2.5*2 cm in size, adjacent to the mass (I). Microscopic findings revealed these lesions were PRNRP and UC, respectively. PCR analysis revealed a KRAS gene mutation (G12D in exon 2) in the PRNRP, while NGS analysis revealed FGFR3 (S249C in exon 7) and KDM6A (Q271Ter in exon 10 and A782Lfs in exon 17) mutations in the UC.ConclusionsWe report here for the first time an extraordinarily rare case of synchronous renal tumors of a PRNRP and UC in the ipsilateral kidney of an RTR. We identified simultaneous KRAS, FGFR3, and KDM6A mutations in two different renal masses in the ipsilateral kidney. Pathologic assessment with comparative molecular analysis of mutational profiles facilitates tumor studies after RT and may be of great value in clinical management strategies.

Landscape of Clinical Resistance Mechanisms to FGFR Inhibitors in FGFR2-Altered Cholangiocarcinoma.

FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

FGFR1 variants contributed to families with tooth agenesis

BackgroundTooth agenesis is a common dental anomaly that can substantially affect both the ability to chew and the esthetic appearance of patients. This study aims to identify possible genetic factors that underlie various forms of tooth agenesis and to investigate the possible molecular mechanisms through which human dental pulp stem cells may play a role in this condition.ResultsUsing whole-exome sequencing of a Han Chinese family with non-syndromic tooth agenesis, a rare mutation in FGFR1 (NM_001174063.2: c.103G > A, p.Gly35Arg) was identified as causative and confirmed by Sanger sequencing. Via GeneMatcher, another family with a known variant (NM_001174063.2: c.1859G > A, p.Arg620Gln) was identified and diagnosed with tooth agenesis and a rare genetic disorder with considerable intrafamilial variability. Fgfr1 is enriched in the ectoderm during early embryonic development of mice and showed sustained low expression during normal embryonic development of Xenopus laevis frogs. Functional studies of the highly conserved missense variant c.103G > A showed deleterious effects. FGFR1 (c.103G > A) was overexpressed compared to wildtype and promoted proliferation while inhibiting apoptosis in HEK293 and human dental pulp stem cells. Moreover, the c.103G > A variant was found to suppress the epithelial-mesenchymal transition. The variant could downregulate ID4 expression and deactivate the TGF-beta signaling pathway by promoting the expression of SMAD6 and SMAD7.ConclusionOur research broadens the mutation spectrum associated with tooth agenesis and enhances understanding of the underlying disease mechanisms of this condition.

SAT329 Case Series Of Patients With FGFR-1 Related Pheochromocytoma With A Focus On Biochemical And Imaging Signatures

Abstract Disclosure: S. Jhawar: None. J. Del Rivero: None. K. Pacak: None. Introduction: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal gland and neural crest, respectively. 40% of cases are associated with germline mutations and 30-40% somatic driver mutations. Here, we present two patients with PCC who were found to have somatic FGFR1 mutation. FGFR signaling is involved in regulation of tissue repair, angiogenesis, and inflammation. Patient 1: A 70-year-old female with a history of resistant hypertension, episodic palpitations and sweating presented with diffuse abdominal pain and chest tightness. She was noted to have a blood pressure of 220/160 mm Hg. CT scan showed a 4.2 x 3.9 cm left adrenal mass, suspicious for malignancy and a <1 cm retroperitoneal mass. Blood work revealed elevated plasma normetanephrine (NM) 3543 pg/mL (normal 0- 145) and metanephrine (MN) 871 pg/mL (normal 0-62). 18F-FDG PET/CT showed a left adrenal mass without abnormal FDG activity. 68Ga-DOTATATE PET/CT was compatible with left PCC (SUVmax 28). 18F-DOPA PET/CT showed left PCC and a small retroperitoneal PGL. Two weeks after initiating phenoxybenzamine, she underwent total left adrenalectomy. Pathology was consistent with PCC. Molecular testing revealed FGFR1 pathogenic somatic mutation (c.1638C>G p.Asn546Lys). Patient 2: A 73-year-old male with known left renal cell carcinoma presented to our institution for management of recurrent right PCC. He was found to have a 3.8 cm right adrenal incidentaloma eight years ago and elevated plasma NM 6.3 nmol/L (normal <0.9), MN 1.5 nmol/L (normal <0.5). He underwent total right adrenalectomy, and the diagnosis of PCC was confirmed on histopathology. Two years later, he developed a 5.1 x 4.7 cm nodular mass in the right adrenal fossa and a <1 cm liver lesion on CT scan. The adrenal lesion showed intense avidity on 68Ga-DOTATATE PET/CT and 18F-DOPA PET/CT and mild avidity on 18FDG-PET/CT. 18F-DOPA PET/CT also showed avidity towards the liver lesion. He was started on doxazosin and underwent laparoscopic right adrenalectomy. Pathology demonstrated PCC. Whole exome sequencing of the tumor showed pathogenic FGFR1 mutation (c.1638C>G, p.Asn546Lys). Discussion: Activating FGFR1 mutations are associated with sporadic PCC. The biochemical phenotype of our patients showed higher levels of NM than MN. The imaging profile revealed that 18F-DOPA PET/CT was more sensitive than 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT as it showed avidity towards other extra-adrenal lesions. Further studies in larger cohorts are needed to enhance understanding of pathogenesis, biochemical and imaging phenotype, which will help tailor the treatment and potential development of novel targeted therapies. Presentation: Saturday, June 17, 2023

OR34-04 Defective FGFR1 Signaling Disrupts Glucose Regulation In Humans: Evidence From Patients With Isolated Hypogonadotropic Hypogonadism Harboring FGFR1 Mutations

Abstract Disclosure: M. Stamou: None. C.J. Chiu: None. S. Jadhav: None. K. Salnikov: None. L. Plummer: None. S.B. Seminara: None. R. Balasubramanian: None. Introduction: Recent studies have shown that antibody-mediated activation of FGFR1 receptor improves metabolic health in rodents, nonhuman primates, and humans. Hence, disruption of the FGFR1 receptor may lead to glucose dysregulation. FGFR1 mutations contribute to 12% of the genetic background of subjects with isolated hypogonadotropic hypogonadism (IHH), a rare Mendelian disease caused by GnRH deficiency. In this study, we examined glucose effectiveness and insulin sensitivity in IHH subjects with rare FGFR1 variants and hypothesized that these subjects will display defective glucose metabolism compared healthy non-carrier controls. Methods: Nine subjects with IHH (7 males and 2 females) with heterozygous rare FGFR1 variants were enrolled [7 subjects with protein truncating variants: p.Q680*, p.R622*, p.Q52*, p.I347Nfs*61, p.R576Pfs*77, p.Q309*, c.1553-2A>G and 2 subjects with deleterious missense variants: p.G97S & p.Y701C. Thirty healthy controls (17 males and 13 females) with no FGFR1 variants were enrolled from the Mass General Brigham Biobank. Medical history, physical exam, laboratory evaluation, a bone density scan, and an intravenous glucose tolerance test (IVGTT) were performed. The area under the curve (AUC) for glucose, insulin, and C-peptide response to 50% dextrose (0.3g/kg, t=0min) and an insulin bolus (0.03u/kg, t=20 min) were estimated for each subject. The glucose effectiveness (Sg), insulin sensitivity (SI), acute insulin response to glucose (AIR), and the disposition index (DI= AIRG*SI) were calculated using the Minimal Model Analysis. Statistical comparisons were performed with t-test and non-parametric Wilcoxon rank sum test. A linear regression model was used to adjust the t-test outcomes for potential confounders. Results: Despite similar BMIs (28 vs. 26.5, p=0.29), IHH FGFR1 positive subjects demonstrated higher % fat mass compared to controls (38 vs. 31, p=0.02). IHH patients were supplemented with sex-steroids at the time of the study. Male patients demonstrated similar testosterone levels compared to controls (385 vs. 451 ng/dL, p=0.52). IHH FGFR1 positive subjects demonstrated a higher AUC for C-peptide compared to controls (167 vs. 92, p=0.001), that remained significant after adjusting for age, gender, BMI, and % fat mass. Non-parametric test showed higher AUC for glucose, insulin, and C-peptide (p 0.01, 0.01 & 0.005 respectively) as well as lower SI (p=0.01) and higher AIRg (p=0.037) in the IHH FGFR1 positive group compared to non-carrier controls. Discussion: This is the first study to evaluate glucose metabolism in humans with naturally occurring FGFR1 variants. IHH FGFR1 positive subjects showed higher insulin response to glucose and lower insulin sensitivity during an IVGTT test compared to the healthy non-carrier controls. These findings confirm that FGFR1 signaling is a key regulator of insulin secretion and action in humans. Presentation: Sunday, June 18, 2023

Comparative Genomic Analysis and Clinical Outcomes of BRAF-mutated Advanced Biliary Tract Cancers.

BRAF mutations are rare in biliary tract cancers (BTC), but are of interest given the recent developments in targeted therapy for BTC. We investigated the clinical outcomes in a cohort of BRAF-mutant advanced BTC treated with first-line chemotherapy. Furthermore, we investigated the genomic landscape of BRAF class I, II, and III mutations in the intrahepatic cholangiocarcinoma (iCCA) subgroup of BTC. We analyzed two nonoverlapping cohorts. We examined the genomic landscape of BRAF-mutated iCCA in a "genomic cohort" [187 class I, 82 class II, 113 class III BRAF mutants and 8,026 wildtype (WT)]. We also analyzed median progression-free survival (PFS) and overall survival (OS) on first-line chemotherapy in a separate multi-institutional "clinical cohort" of patients with BTC (including iCCA and extrahepatic cholangiocarcinoma (eCCA) and gallbladder cancer; 41 class I, 32 class II+III BRAF mutants and 1,042 WT). In the entire BTC clinical cohort, the median PFS was shorter for class I [HR, 2.11 (P < 0.001)] and class II+III [HR, 1.72 (P = 0.007)] as compared with BRAF WT. OS was also shorter in class I [HR, 2.04 (P = 0.011)] and class II+III [HR, 1.86 (P = 0.002)] as compared with BRAF WT. In the iCCA subgroup, class I alterations were mutually exclusive with FGFR2, IDH1/2, ERBB2, and KRAS mutations. Class II+III mutations appear to be mutually exclusive with FGFR2 and KRAS. In BTC, all classes of BRAF mutations are associated with a worse prognosis. BRAF mutations occur in 5% of iCCA subgroup and may be mutually exclusive with other targetable mutations.

Embryonic cranial cartilage defects in the Fgfr3Y367C /+ mouse model of achondroplasia.

Achondroplasia, the most common chondrodysplasia in humans, is caused by one of two gain of function mutations localized in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leading to constitutive activation of FGFR3 and subsequent growth plate cartilage and bone defects. Phenotypic features of achondroplasia include macrocephaly with frontal bossing, midface hypoplasia, disproportionate shortening of the extremities, brachydactyly with trident configuration of the hand, and bowed legs. The condition is defined primarily on postnatal effects on bone and cartilage, and embryonic development of tissues in affected individuals is not well studied. Using the Fgfr3Y367C/+ mouse model of achondroplasia, we investigated the developing chondrocranium and Meckel's cartilage (MC) at embryonic days (E)14.5 and E16.5. Sparse hand annotations of chondrocranial and MC cartilages visualized in phosphotungstic acid enhanced three-dimensional (3D) micro-computed tomography (microCT) images were used to train our automatic deep learning-based 3D segmentation model and produce 3D isosurfaces of the chondrocranium and MC. Using 3D coordinates of landmarks measured on the 3D isosurfaces, we quantified differences in the chondrocranium and MC of Fgfr3Y367C/+ mice relative to those of their unaffected littermates. Statistically significant differences in morphology and growth of the chondrocranium and MC were found, indicating direct effects of this Fgfr3 mutation on embryonic cranial and pharyngeal cartilages, which in turn can secondarily affect cranial dermal bone development. Our results support the suggestion that early therapeutic intervention during cartilage formation may lessen the effects of this condition.

Nested and Large Nested Subtypes of Urothelial Carcinoma of the Upper Urinary Tract: A Multi-institutional Study

Nested urothelial carcinoma (NUC) and large nested urothelial carcinoma (LNUC) of the upper urinary tract are exceedingly rare. This has contributed to the paucity of information regarding their clinicopathological and molecular characteristics. To address this knowledge gap, we explored the largest cohort to date of these rare tumors, comprising resection specimens of 10 LNUC and 7 NUC, from 7 participating institutions. Clinicopathological data were retrieved and documented. Whole exome sequencing and RNA sequencing were performed on the Illumina NovaSeq 6000 sequencer. The data generated were analyzed using the genome analysis toolkit pipeline. Somatic mutations were annotated using funcotator tool to identify pathogenic/likely pathogenic variants. Tumor mutational burden was calculated using python-based “pyTMB” tool. Microsatellite instability analysis was done using MSIsensor2 and the Idylla platform. Differential expression analysis of genes in LNUC and NUC along with mRNA expression–based molecular subtyping was performed by analyzing expression pattern of markers used in The Cancer Genome Atlas subclassification of bladder carcinoma. Both tumor types were more common in older males, were unifocal, and occurred more commonly mixed with minor components of predominantly conventional urothelial carcinoma. Overlying low-grade papillary urothelial carcinoma was significantly more common in LNUC (P = .034). On follow-up (LNUC: median, 10 months; range, 3-84 months; NUC: median, 9 months; range, 2-48 months), LNUC had better clinical outcomes (P = .031). Pathogenic mutations in FGFR3 and PIK3CA were significantly more common in LNUC (P = .049 and P = .044, respectively), with the latter present exclusively in LNUC. Seventy-five percent of the cases showed tumor mutational burden of <10, and all cases were microsatellite-stable. FGFR3 mutations were also more common in low-stage tumors. This study expands on the clinicopathological spectrum of NUC and LNUC of the upper urinary tract and is the first to comprehensively analyze the molecular profile of these tumors, highlighting pathogenic genetic alterations of potential therapeutic and prognostic value.

Molecular characterisation of tumours of the lacrimal apparatus.

Malignant tumours of the lacrimal apparatus are rare and frequently show a poor prognosis, with no clear therapeutic standards. Characterisation of the genetic landscape of these rare tumours is sparse, and therefore therapeutics generally follow those of their common salivary gland counterparts. To further clarify the pathophysiology and discover potential therapeutic targets, we investigated the genetic landscape of eight tumours of the lacrimal apparatus. DNA and RNA sequencing were performed to identify genetic mutations and gene fusions. Immunohistochemistry, fluorescence in-situ hybridisation and reverse transcription-polymerase chain reaction followed by Sanger sequencing were performed to confirm the identified molecular alterations. Genetic alterations were detected in six tumours. Among five adenoid cystic carcinomas (ACC), four had confirmed alterations of MYB or MYBL1 genes, including a MYB::NFIB fusion, a MYBL1::NFIB fusion, a MYB amplification and a novel NFIB::THSD7B fusion. Mutations in genes encoding epigenetic modifiers, as well as NOTCH1, FGFR2 and ATM mutations, were also identified in ACCs. A carcinoma ex pleomorphic adenoma showed TP53 and CIC mutations and an amplification of ERBB2. A transitional cell carcinoma was associated with HPV16 infection. No genetic alteration was found for one adenocarcinoma, not otherwise specified. Our study highlights the variety of molecular alterations associated with lacrimal system tumours and emphasises the importance of molecular testing in these tumours, which can reveal potentially targetable mutations. Our results also reinforce the hypothesis of a common physiopathology of all ACCs, regardless of their primary location.

Clinical and Genomic Landscape of FGFR3-Altered Urothelial Carcinoma and Treatment Outcomes with Erdafitinib: A Real-World Experience.

Erdafitinib is the only FDA-approved targeted therapy for FGFR2/3-altered metastatic urothelial cancer. We characterized the genetic landscape of FGFR-altered urothelial carcinoma and real-world clinical outcomes with erdafitinib, including on-treatment genomic evolution. Prospectively collected clinical data were integrated with institutional genomic data to define the landscape of FGFR2/3-altered urothelial carcinoma. To identify mechanisms of erdafitinib resistance, a subset of patients underwent prospective cell-free (cf) DNA assessment. FGFR3 alterations predictive of erdafitinib sensitivity were identified in 39% (199/504) of patients with non-muscle invasive, 14% (75/526) with muscle-invasive, 43% (81/187) with localized upper tract, and 26% (59/228) with metastatic specimens. One patient had a potentially sensitizing FGFR2 fusion. Among 27 FGFR3-altered cases with a primary tumor and metachronous metastasis, 7 paired specimens (26%) displayed discordant FGFR3 status. Erdafitinib achieved a response rate of 40% but median progression-free and overall survival of only 2.8 and 6.6 months, respectively (n = 32). Dose reductions (38%, 12/32) and interruptions (50%, 16/32) were common. Putative resistance mutations detected in cfDNA involved TP53 (n = 5), AKT1 (n = 1), and second-site FGFR3 mutations (n = 2). FGFR3 mutations are common in urothelial carcinoma, whereas FGFR2 alterations are rare. Discordance of FGFR3 mutational status between primary and metastatic tumors occurs frequently and raises concern over sequencing archival primary tumors to guide patient selection for erdafitinib therapy. Erdafitinib responses were typically brief and dosing was limited by toxicity. FGFR3, AKT1, and TP53 mutations detected in cfDNA represent putative mechanisms of acquired erdafitinib resistance.

Differential outcomes and immune checkpoint inhibitor response among endometrial cancer patients with MLH1 hypermethylation versus MLH1 “Lynch-like” mismatch repair gene mutation

ObjectivesTo identify differential survival outcomes and immune checkpoint inhibitor (ICI) response in MLH1 hypermethylated versus MLH1 mutated (“Lynch-like”) endometrial tumors and determine whether their molecular profiles can elucidate the differential outcomes. Methods1673 mismatch repair deficient endometrial tumors were analyzed by next-generation sequencing and whole transcriptome sequencing (Caris Life Sciences, Phoenix, AZ). PD-L1, ER, and PR were tested by immunohistochemistry and immune cell infiltrates were calculated using MCP-counter. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons. Overall survival (OS) was depicted using Kaplan-Meier survival curves. ResultsThe endometrial cancer cohort comprised 89.2% patients with MLH1 hypermethylated tumors and 10.8% with MLH1 mutated tumors, with median ages of 67 and 60 years, respectively (p < 0.01). Patients with MLH1 hypermethylated tumors had significantly worse OS and trended toward worse OS following ICI treatment than patients with MLH1 mutated tumors. The immune microenvironment of MLH1 hypermethylated relative to MLH1 mutated was characterized by decreased PD-L1 positivity, immune checkpoint gene expression, immune cell infiltration, T cell inflamed scores, and interferon gamma (IFNγ) scores. MLH1 hypermethylation was also associated with decreased mutation rates in TP53 and DNA damage repair genes, but increased rates of JAK1, FGFR2, CCND1, and PTEN mutations, as well as increased ER and PR positivity. ConclusionsEndometrial cancer patients with MLH1 hypermethylation display significantly decreased survival and discrepant immunotherapy responses compared to patients with MLH1 mutated tumors, which was associated with differential mutational profiles, a more immune cold phenotype, and increased ER/PR expression in MLH1 hypermethylated tumors. Providers may consider early transition from single agent ICI to a multi-agent regimen or hormonal therapy for patients with MLH1 hypermethylated tumors.

Clinical and molecular features of 40 Chinese patients with idiopathic hypogonadotropic hypogonadism.

Male idiopathic hypogonadotropic hypogonadism (IHH) is a heterogeneous clinical rare genetic disorder that can be divided into two forms: Kallmann syndrome (KS) and olfactory normal IHH (nIHH). Nearly half of unknown pathogenic genes and related pathogenic mechanisms have yet to be explored. Clinical data of 40 IHH patients (22 KS and 18 nIHH) were retrospectively recorded. All patients were diagnosed at the Department of Endocrinology of Jinling Hospital, Jiangsu Provincial People's Hospital, and the First Affiliated Hospital of the University of Science and Technology of China from 2014 to 2021. The proband genomic DNA (gDNA) was confirmed by whole exome sequencing (WES) and Sanger sequencing. Ten new genetic mutations related to IHH in four families and eight sporadic unrelated IHH patients were identified. The total positive detection rate of 40 patients was 30% (nIHH 8/18 + KS 4/22), and the FGFR1 mutation rate accounted for 7.5% (3/40). Mutation rates of ANOS1, CHD7, and KISS1R were 5% (2/40), respectively. The mutation rates of SEMA3E, PROKR2, and SOX10 were 2.5% (1/40), respectively. After analysis by SIFT and PolyPhen-2 software, all missense mutation sites, such as SEMA3E (p.P323S), CHD7 (p.W1785C), PROKR2 (p.Y223D and p.R298C), were harmful; all nonsense mutation sites, such as FGFR1 (p.R661X) and KISS1R (p.R331X, p.Y103X), analyzed were pathogenic by Mutation Taster software. The comparison of MEGA5 software showed that all the variants had extremely high homology among different species and were extremely conservative in evolution. The study aims to expand the genotype mutation spectrum of IHH and provide evidence for the follow-up clinical treatment and genetic counseling of the disease.

Unraveling the Mystery: Next Generation Sequencing Sheds Light on Neuroblastoma Pathogenesis and Targeted Therapies.

There is considerable interest in the molecular evaluation of solid tumors in pediatric cases. Although clinical trials are in progress for targeted therapies against neuroblastoma (NB), novel therapeutic strategies are needed for high-risk cases that are resistant to therapy. The aim of the present study was to document the specific gene mutations related to targeted therapy in relapsed or refractory NB patients by using next generation sequencing (NGS). The study included 57 NB patients from amongst 1965 neuroblastic cases in Turkey who experienced a recurrence after multi-model therapy. The cases were diagnosed, risk-stratified, and treated according to the classification system from the International Neuroblastoma Risk Group. Single nucleotide variations in 60 genes were investigated using the Pillar Onco/Reveal Multicancer v4 panel and Pillar RNA fusion panel on the Illumina Miniseq platform. ERBB2 I655V was the most frequent mutation and was found in 39.65% of cases. Anaplastic Lymphoma Kinase (ALK) mutations (F1174L, R1275Q, and rare mutations in the tyrosine kinase domain) were detected in 29.3% of cases. Fusion mutations in NTRK1, NTRK3, ROS1, RET, FGFR3, ALK and BRAF were observed in 19.6% of cases. This study presents valuable mutation data for relapsed and refractory NB patients. The high frequency of the ERBB2 I655V mutation may allow further exploration of this mutation as a potential therapeutic target. Rare BRAF mutations may also provide opportunities for targeted therapy. The role of ABL1 mutations in NB should also be explored further.

Genetic Subtypes of Apert Syndrome Are Associated With Differences in Airway Morphology and Early Upper Airway Obstruction.

Apert syndrome is predominantly caused by 2 paternally inherited gain-of-function mutations in the FGFR2 gene, Pro253Arg, and Ser252Trp. Studies comparing phenotypic features between these 2 mutations have established differences in syndactyly severity and incidence of cleft palate. Obstructive sleep apnea can be debilitating in a subset of patients with Apert syndrome, yet is not well understood. This study aims to determine whether FGFR2 mutations impart differential effects on airway physiology and morphology. Patients with Apert syndrome and confirmatory molecular testing were reviewed for polysomnography, nasal endoscopy, microlaryngoscopy and bronchoscopy, and computed tomography imaging. Obstructive apnea-hypopnea index and oxygen saturation nadir, nasal airway volumes, choanal cross-sectional area, and midfacial cephalometric dimensions were compared across mutation types. Twenty-four patients (13 Ser252Trp, 11 Pro253Arg) were included. Severe obstructive sleep apnea (obstructive apnea-hypopnea index>10) occurred in 8 (62%) patients with Ser252Trp mutations compared with 1 (9%) patient with Pro253Arg mutations ( P =0.009). Computed tomography imaging at 1 year of age demonstrated that nasopharyngeal airway volumes were 5302±1076mm 3 in the Ser252Trp group and 6832±1414mm 3 in the Pro253Arg group ( P =0.041). Maxillary length (anterior nasal spine-posterior nasal spine, P =0.026) and basion-anterior nasal spine ( P =0.007) were shorter in patients with Ser252Trp mutations. The findings suggest that the Ser252Trp mutation in Apert syndrome is associated with higher severity obstructive sleep apnea and decreased nasopharyngeal airway volume. Heightened clinical awareness of these associations may inform treatment planning and family counseling.

Transcriptomic Profiling of Upper Tract Urothelial Carcinoma: Bladder Cancer Consensus Classification Relevance, Molecular Heterogeneity, and Differential Immune Signatures

Analyses of large transcriptomics data sets of muscle-invasive bladder cancer (MIBC) have led to a consensus classification. Molecular subtypes of upper tract urothelial carcinomas (UTUCs) are less known. Our objective was to determine the relevance of the consensus classification in UTUCs by characterizing a novel cohort of surgically treated ≥pT1 tumors. Using immunohistochemistry (IHC), subtype markers GATA3-CK5/6-TUBB2B in multiplex, CK20, p16, Ki67, mismatch repair system proteins, and PD-L1 were evaluated. Heterogeneity was assessed morphologically and/or with subtype IHC. FGFR3 mutations were identified by pyrosequencing. We performed 3’RNA sequencing of each tumor, with multisampling in heterogeneous cases. Consensus classes, unsupervised groups, and microenvironment cell abundance were determined using gene expression. Most of the 66 patients were men (77.3%), with pT1 (n = 23, 34.8%) or pT2-4 stage UTUC (n = 43, 65.2%). FGFR3 mutations and mismatch repair–deficient status were identified in 40% and 4.7% of cases, respectively. Consensus subtypes robustly classified UTUCs and reflected intrinsic subgroups. All pT1 tumors were classified as luminal papillary (LumP). Combining our consensus classification results with those of previously published UTUC cohorts, LumP tumors represented 57.2% of ≥pT2 UTUCs, which was significantly higher than MIBCs. Ten patients (15.2%) harbored areas of distinct subtypes. Consensus classes were associated with FGFR3 mutations, stage, morphology, and IHC. The majority of LumP tumors were characterized by low immune infiltration and PD-L1 expression, in particular, if FGFR3 mutated. Our study shows that MIBC consensus classification robustly classified UTUCs and highlighted intratumoral molecular heterogeneity. The proportion of LumP was significantly higher in UTUCs than in MIBCs. Most LumP tumors showed low immune infiltration and PD-L1 expression and high proportion of FGFR3 mutations. These findings suggest differential response to novel therapies between patients with UTUC and those with MIBC.

Urinary tumor DNA MRD analysis to identify responders to neoadjuvant immunotherapy in muscle-invasive bladder cancer.

Bladder preservation is a viable option for some muscle-invasive bladder cancer (MIBC) patients, but an effective noninvasive biomarker test to accurately identify promising candidates is lacking. Here we present the clinical application of a novel tissue-agnostic, urine-based minimal residual disease (MRD) assay in the neoadjuvant setting for personalized disease surveillance and actionable target identification to facilitate bladder-sparing treatment approaches. The urinary tumor DNA (utDNA) analysis was evaluated in an investigator-initiated phase I trial RJBLC-I2N003 in which 20 patients diagnosed with resectable MIBC were treated presurgically with the PD-1 inhibitor toripalimab followed by radical cystectomy (RC). We showed that neoadjuvant toripalimab therapy was feasible, safe, and induced a 40% rate (8/20) of pathological complete response. Longitudinal utDNA profiling outperformed radiographic assessment and conventional biomarkers to predict the pathologic outcome of immune checkpoint blockade. In addition to detecting 3 exceptional responders with molecular MRD-negative status, we identified 7 other individuals characterized for utDNA clearance and 4 harboring FGFR3 mutants, all of whom (60%, 12/20) could have postponed or avoided RC. These findings demonstrate the safety and efficacy of neoadjuvant toripalimab, and suggest the immense potential of noninvasive utDNA MRD testing to guide tailored decision-making with regard to bladder preservation and change the current treatment paradigm for MIBC patients.

3H-3000, AN FGFR2B-SPECIFIC MONOCLONAL ANTIBODY, SHOWS SUPERIOR TUMOR KILLING IN VIVO

Abstract Background and significance Fibroblast growth factor receptor 2b (FGFR2b), a splice isoform of FGFR2, is found to overexpress across multiple cancers types and promotes dysregulated tyrosine kinase activation, resulting in tumor cell proliferation and unchecked malignancy. Cohort studies reveal FGFR2b overexpression and/or amplification in Gastric cancer (~30.2%), squamous non-small cell lung cancer (~20.8%), and endometrial carcinoma (~40%). About 80–85% patients who have advanced HER2-negative gastro-esophageal cancer undergoes approximately 12-14 months of median overall survival (mOS). Thus, there is an urgent need for novel and effective molecular targeted agents. With the recent advancement of mechanistic studies, however, FGFR2b is becoming the optimal target of multiple modalities such as mAb, bispecifics, or antibody-drug conjugate. Bemarituzumab, a FGFR2b blocking antibody, developed by Five Prime clinically demonstrates the safety of FGFR2b target and exhibits preliminary efficacy in advanced GE/GEJ adenocarcinoma patients with overexpression of FGFR2 in at least 5% or 10% of tumor cells. 3H Pharmaceuticals developed a FGFR2b-specific mAb, 3H-3000, of high-affinity, differentiated epitope and enhanced ADCC effect for FGFR2b-overexpressing gastric cancer or other FGFR2b-associated cancer types. In the meantime, we will also explore the versatility of this mAb in other format and in the combination with small molecules that will bring about a wider therapeutic spectrum. Method and Result 3H-3000 is a humanized IgG1kappa antibody with a single digit nM affinity for FGFR2b and no binding to other FGFR2 isoforms. 3H-3000 shows potent proliferation-inhibitive effects on FGFR2b-overexpressing human tumor cells. In gastric cancer cell line SNU16, 3H-3000 can fully inhibit FGF7-induced phosphorylation of FGFR2b and SNU16 proliferation in vitro. On top of signaling blocking, we strongly believe efficacy of antibody-dependent cell cytotoxicity (ADCC) is another viable mechanism for growth factor-targeting therapy. Therefore, 3H-3000 was further engineered with a 20-fold ADCC enhancement which is validated through a CD16-F158 variant-based reporter system. In FGFR2b-overexpressing human gastric cancer SNU16 or OCUM-2M xenograft model, 3H-3000 was shown to inhibit tumor growth and even induce regression, which is rather unexpected given the relative low expression of FGFR2b on SNU16 and none of the competitors exhibit similar phenomenon. With potent efficacy of tumor inhibition and killing, and excellent biophysical characteristics and developability of 3H-3000, we expect to push the molecule to clinical development in the middle of 2024. Conclusion 3H-3000 is a potent FGFR2b blocker with well-defined ADCC efficacy enhancement. It demonstrates strong blocking activity of FGFR2 signaling, strikingly enhanced ADCC activity in vitro and potent efficacy of tumor inhibition in vivo. These data strongly support its clinical development in FGFR2b-overexpressing cancers. In parallel, 3H Pharmaceuticals are also developing FGFR2- selectively tyrosine kinase inhibitors targeting FGFR2 mutations or fusion. We propose a combination of small molecule FGFR2 inhibitors and mAb of FGFR2b specificity will significantly broaden the therapeutic spectrum of heterogenous tumors where mechanism is centered around FGFR2b alteration including mutation, fusion, and overexpression.

CEREBELLAR DIFFUSE MIDLINE GLIOMA, H3K27M ALTERED IN A FIFTY-FIVE-YEAR-OLD PATIENT

Abstract H3 K27M mutations were first identified in Pediatric Diffuse Intrinsic Pontine Glioma and are now recognized in gliomas occurring in other midline locations such as the thalamus, spinal cord and cerebellum. These gliomas were renamed to Diffuse Midline Glioma (DMG) H3 K27-altered in the 2021 WHO classification due to the discovery of alternate mechanisms of H3 K27 loss. These gliomas are more commonly found in children but also occur in adolescents and young adults. We present the case of a 55- year-old woman who presented with a 2.5-month history of nausea, vomiting and gait impairment. Imaging revealed a partially cystic, enhancing lesion in the midline of the cerebellum. She had a gross total resection with pathology revealing Diffuse Midline Glioma, H3K27 altered, subgroup DMG, H3 wildtype with EZHIP over-expression, WHO grade 4. The patient completed chemoradiation with temozolomide with plans to enroll in a clinical trial upon progression. However due to loss of ambulation and function she was admitted to hospice. The patient survived 24 months after her surgery, a period longer than the reported median overall survival in DMG with EZIP overexpression which is 16 months. This case illustrates the importance of considering DMG H3K27- altered glioma in adults older than 40 years who present with infiltrative IDH wildtype glioma in midline locations. Recognition of this diagnosis may allow enrollment in clinical trials and should prompt molecular testing for concurrent targetable alterations in the RAS-MAPK-pathway (e.g., mutations in BRAF V600E, FGFR1, NF1 and NTRK) and PI3K-mTOR pathway (e.g., PIK3CA mutation).