Activating Point Mutations Research Articles

The value of ACR, European, Korean, and ATA ultrasound risk stratification systems combined with RAS mutations for detecting thyroid carcinoma in cytologically indeterminate and suspicious for malignancy thyroid nodules.

The aim of this study was to evaluate the diagnostic value of four commonly utilized ultrasound (US) RSSs, namely, the American College of Radiology [ACR], European [EU], Korean [K] TI-RADSs and American Thyroid Association [ATA] US-based RSS criteria, in combination with activating point mutations of the RAS genes (NRAS, HRAS, and KRAS) for detection of thyroid carcinoma in cytologically indeterminate and suspicious for malignancy thyroid nodules. We retrospectively analyzed cytologically indeterminate and suspicious for malignancy thyroid nodules which underwent US, molecular testing and surgery between September 1, 2018, and December 31, 2023. Receiver operating characteristic (ROC) curves were generated, and the area under the curve (AUC, 95% confidence interval [CI]) was calculated. A total of 100 cytologically indeterminate and 24 suspicious for malignancy thyroid nodules were analyzed. Compared to the four US-based RSSs alone, the diagnostic value of the four US-based RSSs combined with RAS mutations did not significantly improved (cytologically indeterminate, AUC [95% CI] 0.6 [0.5-0.7] and 0.6 [0.5-0.7], respectively, p = 0.70; cytologically suspicious for malignancy, AUC [95% CI] 0.7 [0.5-0.9] and 0.8 [0.6-0.9], respectively, p = 0.23). The diagnostic value of the four main US-based RSSs (ACR, EU, K, and ATA) was not improved in conjunction with the evaluation of RAS mutations for preoperative risk stratification of cytologically indeterminate thyroid nodules. In cytologically indeterminate nodules categorized according to US-based RSSs, isolated RAS positivity does not reliably distinguish between benignity and malignancy.

Activating Point Mutations in the MET Kinase Domain Represent a Unique Molecular Subset of Lung Cancer and Other Malignancies Targetable with MET Inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas. Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ∼0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors. Significance: The identification of targetable genomic subsets of cancer has revolutionized precision oncology and offers patients treatments with more selective and effective agents. Here, we demonstrate that activating, oncogenic MET tyrosine kinase domain mutations are found across a diversity of cancer types and are responsive to MET tyrosine kinase inhibitors.

BRAF Mutated and Morphologically Spitzoid Tumors, a Subgroup of Melanocytic Neoplasms Difficult to Distinguish From True Spitz Neoplasms.

Drivers of Spitz neoplasms include activating point mutations in HRAS and Spitz-associated genomic fusions. It has become evident that some BRAF -mutated melanocytic neoplasms can morphologically mimic Spitz tumors (STs). These have been termed BRAF mutated and morphologically spitzoid (BAMS). In this study, 17 experts from the International Melanoma Pathology Study Group assessed 54 cases which included 40 BAMS and 14 true STs. The participants reviewed the cases blinded to the genomic data and selected among several diagnostic options, including BAMS, ST, melanoma, and other. A total of 38% of all diagnostic selections in the BAMS cases were for BAMS, whereas 32% were for ST. In 22 of the BAMS cases, the favored diagnosis was BAMS, whereas in 17 of the BAMS cases, the favored diagnosis was ST. Among the 20 cases in the total group of 54 with the highest number of votes for ST, half were BAMS. Of BAMS, 75% had a number of votes for ST that was within the SD of votes for ST seen among true ST cases. There was poor interobserver agreement for the precise diagnosis of the BAMS (kappa = 0.16) but good agreement that these cases were not melanoma (kappa = 0.7). BAMS nevi/tumors can closely mimic Spitz neoplasms. Expert melanoma pathologists in this study favored a diagnosis of ST in nearly half of the BAMS cases. There are BAMS cases that even experts cannot morphologically distinguish from true Spitz neoplasms.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4752.

Amplification of Mutant NRAS in Melanocytic Tumors With Features of Spitz Tumors

NRAS activating mutations are prevalent in melanocytic neoplasia, occurring in a subset of common acquired melanocytic nevi and ∼30% of cutaneous melanomas. In this study, we described a cohort of 7 distinctive melanocytic tumors characterized by activating point mutations in codon 61 of NRAS with amplification of the mutant NRAS allele and shared clinicopathologic features. These tumors occurred predominantly in younger patients, with a median age of 20 years (range, 6-56 years). They presented as papules on the helix of the ear (4 cases) or extremities (3 cases). Microscopically, the tumors were cellular, relatively well-circumscribed, compound, or intradermal proliferations. The tumor cells often extended into the deep reticular dermis and involved the superficial subcutaneous fat in some cases. The melanocytes were epithelioid to spindled with moderate amounts of cytoplasm and conspicuous nucleoli. They were arranged in short plexiform fascicles, nests, and cords. Some cases had occasional pleomorphic and multinucleated melanocytes. Rare dermal mitotic figures were present in all cases. The dermis contained thick collagen bundles and minimal solar elastosis. Follow-up data were available for 5 patients, with a median period of 4.2 years (range, 1-9 years), during which no recurrences or metastases were reported. Our series highlights a clinicopathologically and molecularly distinctive subset of NRAS-mutated tumors with amplification of the mutant NRAS allele.

ROS-Driven Structural and Functional Fibrinogen Modifications Are Reverted By JAK Inhibition in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are bone marrow diseases characterized by excess clonal hematopoiesis resulting in elevated peripheral blood counts. Subtypes include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They share clinical features (e.g., splenomegaly, thrombotic complications, risk of leukemic transformation), and a common molecular basis: the dysregulation of the JAK-STAT pathway, which confers a hypersensitivity of hematopoietic stem cells (HSC) to the action of growth factors and cytokines, and, as a result, an overproduction of blood cells. Genetic analysis of patients with MPNs revealed that >95% of patients with PV and about 50% of patients with ET or PMF carry an activating point mutation in the JAK2 gene (JAK2-V617F). Despite clinical evidence indicating a higher susceptibility of MPN patients to develop thrombotic disease, the underlying pathogenetic mechanisms are not fully understood. It appears that thrombosis is the result of a severe inflammatory response associated to remarkable oxidative stress in the presence of endothelial dysfunction and a procoagulant environment. Hypercoagulability is a consequence of disrupted hemostasis and fibrinolysis due to damage of the endothelium and defective fibrinolysis. Evidence suggests that overproduction of ROS and a deprived antioxidant system play a major role in the pathogenesis of MPN, as well as in the resistance to tyrosine-kinase inhibitor therapy. Moreover, systemic inflammation is thought to be the main trigger of thrombosis and seems to be mainly mediated by pro-inflammatory cytokines along with endothelial cell dysfunction. Recent studies have provided evidence that fibrinogen plays a multifaceted role in inflammatory responses and autoimmunity. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with integrins, the leukocyte cell surface adhesion receptors expressed on neutrophils, monocytes, macrophages, and several subsets of lymphocytes. In MPN patients, endothelial cell dysfunction, increased reactive oxygen species (ROS) production, and proinflammatory cytokines have been reported, together with impaired fibrinolysis. We also investigated the potential role of oxidative stress and inflammation in the promotion of thrombosis. In order to study the mechanisms of inflammation-induced thrombosis in MPN patients, the aim of the present study was to investigate the role of oxidation-induced structural and functional fibrinogen modifications in MPN patients. The eventual counteracting effects of ruxolitinib, a first-in-class JAK inhibitor, were also assessed. 40 MPN patients and 40 age- and sex-matched healthy control subjects were included in the study. We measured specific oxidative stress markers andtheir modifications in plasma, and functional and structural parameters in purified fibrinogen fractions. Marked signs of oxidative stress in plasma (P<0.01 versus controls) and, correspondingly, an increased extent of fibrinogen oxidation (P<0.01 versus controls) were observed in patients. Furthermore, fibrinogen fractions purified from MPN patients exhibited significantly reduced clotting ability and decreased susceptibility to plasmin-induced lysis (P<0.01 versus controls). Alterations in fibrinogen secondary structure, as suggested by circular dichroism spectroscopy, and in fibrin clot architecture were also identified. Importantly, clot structure obtained by stimulated emission depletion (STED) super-resolution microscopy indicated modifications in fiber diameter and clot porosity in MPN patients (P<0.01 versus controls). In patient samples, fibrinogen clot analysis and oxidative-dependent changes reveal novel structural and functional fibrinogen modifications which may favor thrombotic complications in MPNs. In patients receiving ruxolitinib, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features, suggesting a potential impactof ruxolitinib on cardiovascular prevention in MPNs.

XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS.

Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRASG12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRASG12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRASG12D -mutated pre-B ALL.

IGF1R Contributes to Cell Proliferation in ALK-Mutated Neuroblastoma with Preference for Activating the PI3K-AKT Signaling Pathway.

Aberrant activation of anaplastic lymphoma kinase (ALK) by activating point mutation or amplification drives 5-12% of neuroblastoma (NB). Previous work has identified the involvement of the insulin-like growth factor 1 receptor (IGF1R) receptor tyrosine kinase (RTK) in a wide range of cancers. We show here that many NB cell lines exhibit IGF1R activity, and that IGF1R inhibition led to decreased cell proliferation to varying degrees in ALK-driven NB cells. Furthermore, combined inhibition of ALK and IGF1R resulted in synergistic anti-proliferation effects, in particular in ALK-mutated NB cells. Mechanistically, both ALK and IGF1R contribute significantly to the activation of downstream PI3K-AKT and RAS-MAPK signaling pathways in ALK-mutated NB cells. However, these two RTKs employ a differential repertoire of adaptor proteins to mediate downstream signaling effects. We show here that ALK signaling led to activation of the RAS-MAPK pathway by preferentially phosphorylating the adaptor proteins GAB1, GAB2, and FRS2, while IGF1R signaling preferentially phosphorylated IRS2, promoting activation of the PI3K-AKT pathway. Together, these findings reveal a potentially important role of the IGF1R RTK in ALK-mutated NB and that co-targeting of ALK and IGF1R may be advantageous in clinical treatment of ALK-mutated NB patients.

Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma

Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.

Abstract B032: KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133

Abstract Introduction: KRASG12D is the most common KRAS mutation in pancreatic ductal adenocarcinoma (PDAC). It is well known that activating point mutations in one allele are sufficient for tumor growth in patients harboring KRASG12D mutations and it is challenging to target this mutation with effective KRASG12D inhibitors. Driver gene amplification is a known mechanism of acquired resistance to therapy in pancreatic cancers. Although other genetic features, such as KRAS gene copy number variations (CNV) and mutant allele fractions (MAF), have been observed, their influence on response to treatment remains unclear. Current discovery revealed that MRTX1133 is a potent, non-covalent and high-affinity small molecule that selectively targets KRASG12D. Therefore, with the efforts underway to develop effective therapy response using the KRASG12D-targeted drug, MRTX1133, it is paramount to improve our understanding regarding the role of KRAS CNV/MAF in PDAC. Methods: Using quantitative digital PCR (DPCR), we first measured the absolute copy number of KRAS wild type (WT), and mutant type (MT) and we then calculated MAF by formula: MAF = MT allele numbers/(MT+WT allele numbers) in DNA from various KRASG12D mutant PDAC cell lines and PDX-derived primary cell lines. 3D viability and cell signaling assays determined sensitivity to MRTX1133 in established and PDX-derived primary cell lines in vitro. We also assessed the representative KRAS gene CNV of those cell lines by TaqMan real time PCR. DPCR was also utilized to determine the MAF in PDX-derived tissues. Results: We found a trend in response to MRTX1133 in KRASG12D cell lines based on their MAF and representative KRAS gene CNV. Resistant, and sensitive KRASG12D cell lines have average MAF of 75.23%, and 52.81% respectively. Most of the resistant cells have higher KRAS gene copy number gain (6-8) compared to sensitive cells (3-4). Interestingly, our data suggests that higher MAF (>53%) and gene copy numbers (>4) may predict a resistant phenotype in KRASG12D cells to MRTX1133. Mechanistically, MRTX1133 inhibits ERK1/2 signaling and increases apoptosis in sensitive cells. Representative sensitive and resistant cell lines are further evaluated for any changes in baseline and MRTX1133-induced MAF and copy number alterations by DPCR and TaqMan real time PCR. However, the results indicated that there are no significant differences in MAF and KRAS CNV in the sensitive and resistant cells on pre and post treatment of MRTX1133. We further extended our study by evaluating the MAF targeting KRASG12D in various PDX-derived tissues by quantitative DPCR. With exception to a few cases, most of the analyzed PDX tissues contained balanced MAF (~50%), which may show sensitivity to MRTX1133 according to our findings. Conclusions: Our current observations indicate 1) KRAS CN gain and MAF increase may appear to play major roles in the progression of a resistant phenotype 2) KRAS CNV and MAF status may contribute to criteria to select PDAC patients who are most likely to benefit from therapy with MRTX1133. Citation Format: Bhaswati Sarcar, Alexandra Tassielli, Liang Wang, Ruifan Dai, Meagan D. Read, Francisca Beato, Dae W. Kim, Pamela J. Hodul, Jennifer B. Permuth, Eric B. Haura, Mokenge P. Malafa, Jason B. Fleming. KRAS copy number variation and mutant allele fractions predict in vitro response of PDX-derived human pancreatic cancer cell lines to KRASG12D inhibitor MRTX1133 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B032.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

Discovery of a Potent and Selective Targeted NSD2 Degrader for the Reduction of H3K36me2.

Nuclear receptor-binding SET domain-containing 2 (NSD2) plays important roles in gene regulation, largely through its ability to dimethylate lysine 36 of histone 3 (H3K36me2). Despite aberrant activity of NSD2 reported in numerous cancers, efforts to selectively inhibit the catalytic activity of this protein with small molecules have been unsuccessful to date. Here, we report the development of UNC8153, a novel NSD2-targeted degrader that potently and selectively reduces the cellular levels of both NSD2 protein and the H3K36me2 chromatin mark. UNC8153 contains a simple warhead that confers proteasome-dependent degradation of NSD2 through a novel mechanism. Importantly, UNC8153-mediated reduction of H3K36me2 through the degradation of NSD2 results in the downregulation of pathological phenotypes in multiple myeloma cells including mild antiproliferative effects in MM1.S cells containing an activating point mutation and antiadhesive effects in KMS11 cells harboring the t(4;14) translocation that upregulates NSD2 expression.

Hyponatraemia – diagnostic difficulties in 2.5-year-old boy with nephrogenic syndrome of inappropriate antidiuresis. Case report

Normal sodium levels in the extracellular fluid are in the range of 135–145 mmol/L. Low serum sodium (hyponatraemia) is a common water and electrolyte balance disorder. Dehydration and overhydration are the most common causes of hyponatraemia in children. This paper describes a case of a 2.5-year-old boy admitted to the paediatric ward due to pneumonia. The child showed reluctance to fluid intake, hypoosmolar hyponatraemia with hypouricaemia, and increased urinary sodium excretion in the absence of abnormalities in hydration status. He had a family history of persistent hyponatraemia and hypertension in his grandfather. After excluding pituitary, thyroid, adrenal cortex pathologies, kidney disfunction, and proliferative processes in the differential diagnosis, cerebral salt wasting syndrome, the syndrome of inappropriate secretion of antidiuretic hormone and the renal-related syndrome of inappropriate antidiuresis were taken into account. The aim of the study was to discuss the differential diagnosis of chronic euvolemic hyponatraemia and to draw attention to the need for genetic testing for the nephrogenic syndrome of inappropriate antidiuretic hormone secretion caused by activating point mutations of the vasopressin 2 receptor gene (V2R).

PSAT228 Hereditary Hypophosphatemic Rickets: Management in Adults

Abstract Introduction Hereditary Hypophosphatemic Rickets (HHR) is a group of rare inherited disorders consisting of X-linked hypophosphatemic rickets (XLHR, prevalence 1 in 20,000 births), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR). XLHR is caused by loss of function mutation of the PHEX gene, ARHR is caused by loss of function mutation of the DMP1/ENPP1 gene and ADHR is caused by activating point mutations in FGF23. These mutations result in renal phosphate wasting, hypophosphatemia, and impaired bone mineralization. It presents in childhood as skeletal deformities, abnormal gait, skeletal pain, dental abnormalities, and stunted growth. Adults have osteomalacia-related pain, fractures, and arthritis. The diagnosis is made by findings consistent with rickets and normal serum calcium, normal 25-hydroxy vitamin D, low-normal 1,25-dihydroxy vitamin D, elevated alkaline phosphatase activity, and low phosphate levels. Urine studies show low urine calcium and decreased tubular maximum reabsorption of phosphate, confirming renal wasting of phosphate. The goal of treatment in adults is to reduce pain, prevent fractures, enhance mobility, and decrease osteomalacia. Phosphate and vitamin D supplementations are used with close monitoring as it is associated with severe adverse events. It can cause hypercalcemia leading to nephrocalcinosis, nephrolithiasis, chronic kidney disease, and hyperparathyroidism. There is no evidence that the treatment affects long-term skeletal complications. Burosumab, a monoclonal antibody that inhibits FGF23, is approved for the treatment of XLHR. No studies are available about the use of Burosumab in ADHR or ARHR. Case Presentation A 18-year-old male with past medical history of hypophosphatemic rickets with multiple fractures, on oral phosphate tablets and calcitriol, presented to the Endocrinology clinic to establish care. He had been following Pediatric Endocrinology in the past. His blood work showed normal calcium, low phosphate of 1.6 mg/dl (normal: 2.5-4.5 mg/dl), vitamin D 25 hydroxy level of 26 ng/ml (normal: 20-100 ng/ml), vitamin D 1,25-dihydroxy level of 25 pg/ml (normal: 30-80 pg/ml), intact parathyroid hormone level of 549 pg/ml (normal: 15-65 pg/ml), and alkaline phosphatase of 438 U/l (normal: 40-150 U/l). Urine calcium was low and urine phosphorus was elevated with phosphate/creatinine ratio more than 2000 (normal: 60-962), consistent with renal phosphate wasting. Genetic testing in the past was negative for PHEX gene mutation, so he was not a candidate for Burosumab. Conclusion There is limited knowledge about Hereditary Hypophosphatemic Rickets, especially ADHR and ARHR due to limited cases (less than 100 cases reported so far). The current standard of treatment of HHR is sub-optimal and associated with adverse effects. Burosumab has not been tested in ADHR or ARHR, so it is unclear if it will be beneficial in this subgroup. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

RF11 | PSAT273 RET Gene Fusion In a Putative Case of Medullary Thyroid Cancer

Abstract Background Oncogenic RET gene fusions have been identified in non-small cell lung cancer and non-medullary thyroid cancers, whereas RET point mutations are the key genetic finding in both inherited and sporadic medullary thyroid cancer (MTC). The presence of a RET gene fusion provides a target for selective RET inhibitor drugs. We describe an unusual patient with suspected MTC and a positive RET gene fusion, with excellent response to targeted therapy. Clinical Case A 50-year-old male presented to our institution with cough, fatigue, heat intolerance, and flushing. His only remarkable history was stage IIIBS Hodgkin lymphoma treated with chemotherapy, in remission for approximately 6 years. CT chest revealed new bulky mediastinal and hilar adenopathy with bulky cervical nodes. VATS biopsy of a mediastinal node showed metastatic neuroendocrine neoplasm with plasmacytoid features that was immunoreactive for calcitonin, CEA and cytokeratin, but negative for TTF1 and PAX-8. The differential diagnosis included MTC or metastatic atypical carcinoid tumor. Next Generation Sequencing was negative, including RET and RAS point mutations. Subsequent testing for re-arrangements showed a KIF5B-RET gene fusion, an oncogene identified in lung adenocarcinoma. Biochemical findings showed a calcitonin level of 16,033 (normal < 10 pg/mL) and CEA 6.9 (normal < 4.7 ng/mL). Chromogranin A was 146.6 (normal < 101.16 ng/mL). ACTH and cortisol were normal. Neck US revealed a calcified 5 mm right upper pole thyroid nodule and extensive bilateral cervical nodes. A provisional diagnosis was made of MTC. Vandetanib was started. Two months later, he reported worsening orthopnea, diarrhea, and weight loss. CT chest showed marked worsening with enlarging mediastinal hilar and supraclavicular adenopathy with new moderate sized pericardial and pleural effusion. Vandetanib was discontinued and Selpercatinib started, following FDA approval. He reported prompt resolution of dyspnea and diarrhea and improved weight and energy. Calcitonin declined to <2 pg/mL, and CEA to1.6 ng/mL four months into treatment. CT neck showed marked shrinkage of lymph nodes with a persistent calcified thyroid nodule. CT chest showed marked shrinkage of mediastinal and hilar masses, with resolution of bronchial narrowing and right pleural effusion. Nine months after presentation, there were no chest symptoms and biochemical markers and imaging remained stable. Conclusion Approximately 25% of MTC tumors lack activating point mutations in RET or RAS genes. Analysis of actionable fusion mutations should be considered if there is metastatic disease. High serum calcitonin levels are not pathognomonic for MTC and tumor histology may overlap with neuroendocrine lung cancers, creating a diagnostic challenge. Depending on whether this patient's tumor originated in the thyroid gland, it may represent an unusual case of RET gene fusion in MTC. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m.

Ezh2Y641F mutations co-operate with Stat3 to regulate MHC class I antigen processing and alter the tumor immune response in melanoma.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2, a chromatin modifying complex, which mediates methylation of lysine 27 on histone 3 (H3K27me3), a repressive chromatin mark. Genetic alterations in EZH2 in melanoma include amplifications and activating point mutations at tyrosine 641 (Y641) whose underlying oncogenic mechanisms remain largely unknown. Here, we found that expression of Ezh2Y641F causes upregulation of a subset of interferon-regulated genes in melanoma cells. Upregulation of these genes was not a direct effect of changes in H3K27me3, but via a non-canonical interaction between Ezh2 and Signal Transducer and Activator of Transcription 3 (Stat3). Ezh2 and Stat3 together function as transcriptional activators to mediate gene activation of numerous genes, including MHC Class 1b antigen processing genes. Furthermore, expression of Stat3 is required to maintain an anti-tumor immune response in Ezh2Y641F melanomas and to prevent melanoma progression and recurrence.

BRAFV600E Mutation in Papillary Carcinoma Thyroid

Background: Thyroid malignancies comprise 1 % of total human malignancies, Papillary Thyroid Carcinoma (PTC) is the most common malignant tumour of thyroid cancers. It is estimated that 80 % of thyroid malignancies are comprised of papillary carcinoma thyroid, which shows increased incidence in women. The most important etiology of PTC is mutation of RET/RAS/BRAF which activates MAPK signaling pathway. The BRAF V600E activating point mutation appears to be highly specific for PTC.
 Methods: It is cross sectional study of 56 cases studied in the Government Medical college, Thiruvananthapuram, Kerala. Consecutive cases of histo-pathologically proven cases were selected and Immunohistochemical (IHC) studies for BRAFV600E were conducted, and results were recorded.
 Result: Our study showed female preponderance and equal distribution of cases above and below 45 years of age. Size of the primary tumour was more than 1cm for majority of cases. In our study out of 56 cases studied 45 patients showed BRAFV600E expression comprising of 80% positivity. A 44% of the total cases studied showed lymph node metastasis.
 Conclusion: We conclude in our study that BRAFV600E mutation is most common mutation in primary PTC patients. BRAFV600E expression most seen in elderly patients and shows strong association with lymph node metastasis. IHC assay is a reliable method to detect BRAFV600E mutation in PTC and can be used as alternative to molecular methods on routine practice

Selpercatinib in RET-fusion positive metastatic non-small cell lung cancer: achievements and gray areas

ABSTRACT Introduction Selpercatinib is a RET selective tyrosine kinase inhibitor with nanomolar potency against diverse RET alterations, including fusions, activating point mutations, and acquired resistance mutations. Rearranged during transfection (RET) gene is a validated target in non-small-cell lung cancer (NSCLC). Selpercatinib is currently approved for adult patients with metastatic RET fusion-positive NSCLC. Areas covered This review summarizes the efficacy and safety data of selpercatinib in the treatment landscape of RET fusion-positive NSCLC. Expert opinion Globally considered, selpercatinib is an optimal treatment choice, in terms of both (systemic and intracranial) efficacy and safety, in patients affected by advanced NSCLC harboring RET fusions as a driver mechanism. Future challenges include the identification of the most appropriate placement for selpercatinib in the treatment algorithm of RET fusion-positive NSCLC (including early stages), the clarification of resistance mechanisms, as well as of its role in EGFR-mutant NSCLC undergoing progression during osimertinib driven by RET alterations.

Abstract 1038: Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer

Abstract Introduction: In pancreatic ductal adenocarcinoma (PDAC), low patient survival rate remains a problem. The activating point mutation of KRAS on codon-12 is present in 70-95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis; recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both PDAC and stromal cells, which modulate tumor microenvironment and metastasis. Therefore, we have developed a novel combination therapy for PDAC by targeting mutated KRASG12D and Gal-1 to target both proliferation and metastasis in PDAC. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a mouse model. Results: Our results demonstrate optimal particle size/zeta potential of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. It inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility and stability suggesting its potential of silencing KRAS without being toxic to the body. The formulation efficiently exhibited KRasG12D silencing and inhibited tumor growth and metastasis in nude mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Martinez Bulnes, Poornima Shaji, Nirnoy Dan, Melida Cantu, Shabnam Malik, Stephen Behrman, Swathi Holla, Murali Mohan Yallapu, Meena Jaggi, Subhash C. Chauhan, Sheema Khan. Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1038.

Identification of an optimal circulating tumor DNA (ctDNA) shedding threshold to detect actionable driver mutations in colorectal and pancreatic adenocarcinoma.

3571 Background: In colorectal cancer (CRC), mutations in KRAS, NRAS, and BRAF predict resistance to anti-EGFR therapies. In pancreatic ductal adenocarcinoma (PDAC), ~90% of patients harbor KRAS mutations, while KRAS wild-type tumors often have clinically actionable fusion alterations. Thus, in both cancers, the accurate ascertainment of RAS and BRAF driver status is essential. Sequencing of cell-free DNA (cfDNA) from plasma allows convenient assessment of a tumor’s molecular profile, but sensitivity can be limited by low ctDNA shedding. We sought to establish a ctDNA shedding threshold at which actionable driver mutations can be reliably detected. Methods: Molecular reports and matched clinical data were obtained from the Duke Molecular Registry of Tumors and the SCRUM-Japan GOZILA and GI-SCREEN. CRC or PDAC patients with a pathogenic KRAS, NRAS, or BRAF activating point mutation (“driver”) present on tissue next-generation sequencing (NGS) assays and who also had cfDNA assay available were included. Tissue NGS included Foundation One CDx and Oncomine Comprehensive Assay. Guardant 360 (G360) was the sole plasma cfDNA assay. 131 CRC and 24 PDAC cases with 189 total G360 assays met criteria and were included. Samples were analyzed according to detection of the driver mutation and the maximum mutant allele frequency (MAF) of non-driver mutations on G360. An optimal cut-point for max MAF was explored among the CRC and PDAC patients using a maximally selected Wilcoxon rank statistic method. Results: 76.8% of driver mutations were in KRAS, 22.6% in BRAF, and 1.9% in NRAS with an overlap of 1 BRAF and 1 NRAS mutation with a KRAS mutation. Overall sensitivity of G360 for drivers was 83.0% for CRC and 54.2% for PDAC. No variants were detected on G360 in 9.1% of CRC and 37.5% of PDAC. Sensitivity for driver mutations increased with higher maximum non-driver MAF, with MAF > 1% predicting sensitivity > 98% (Table). Optimal cut-point analysis identified MAF of > 0.34% (p < 0.0001), above which the driver was identified in 97% of patients and below which only 27%. Conclusions: In our study, non-driver MAF > 1% on cfDNA NGS predicts high sensitivity for RAS and BRAF mutations and thus is adequate to guide clinical decisions such as anti-EGFR therapy in CRC, evaluation for fusions in PDAC, and validity in clinical trials. MAF ≤0.34% is a clear threshold to consider an assay inadequate and thus seek alternative testing. Sensitivity rises for MAF between 0.35 and 1% but will require greater patient numbers to establish clinically relevant sensitivity thresholds. These results will be updated with additional data for final presentation. [Table: see text]