Additional Gene Mutations Research Articles

Prospective Evaluation of Sorafenib Combined with Chemotherapy in Newly Diagnosed Adult Core-Binding Factor Acute Myeloid Leukemia: An Open-Label, Randomized Controlled, Multicenter Phase II Trial

Objective Core-binding factor acute myeloid leukemia (CBF-AML) includes AML with t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) chromosomal rearrangements, leading to the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively. Although CBF-AML is considered to have a favorable prognosis relative to other AML subtypes, up to 40% of these patients experience relapse. Recent studies demonstrate achievement of complete molecular remission (CMR) at the end of treatment significantly associated with a reduced risk of relapse. A high frequency of mutations and/or high expression of KIT gene is observed in CBF-AML. Sorafenib is a first-generation type-II multitargeted tyrosine kinase receptor inhibitor (TKI) that suppresses various signaling pathways associated with the development of AML, such as RTK (FLT3, c-KIT). Thus the purpose of this study is to evaluate the safety and efficacy of sorafenib combined with chemotherapy in adult newly diagnosed CBF-AML. Methods Patients were randomly assigned (1:1) to control group (chemotherapy alone) or sorafenib group (sorafenib combined with chemotherapy). For control group, patients received one cycle of induction therapy with idarubicin (12 mg/m² on days 1-3) plus cytarabine (100 mg/m² on days 1-7), followed by one cycle of idarubicin (8 mg/m² on days 1-3) plus cytarabine (2 g/m² q12h on days 1-3) and two cycles of high-dose cytarabine consolidation therapy (2 g/m² q12h on days 1-3). For sorafenib group, patients were treated with chemotherapy plus sorafenib at 400 mg twice daily on days 8-21 for induction cycle, and on days 1-21 for each consolidation, and as maintenance for 12 months. Allogeneic or autologous HSCT were implemented based on the measurable residual disease (MRD) level and donor availability after 4 cycles of therapy. MRD levels were serially monitored for RUNX1-RUNX1T1 or CBFB-MYH11 transcripts by real-time quantitative polymerase chain reaction. Results were expressed as a [fusion gene/ABL1] ×100 transcript ratio. Major molecular response (MMR) and complete molecular remission (CMR) are defined as transcript ratio < 0.1% and 0.001%, respectively. The primary endpoint was CMR after 4 cycles of therapy in bone marrow (BM). Results Between January 2020 and March 2022, 64 patients were enrolled and randomly assigned to sorafenib group (n=32) or control group (n=32). There is no significant difference between the two groups about age, gender, CBF subtype, BM blast percentage, additional chromosome abnormalities and gene mutations at diagnosis. The hematological complete remission in the sorafenib and control group were 96.9% and 94.1%, respectively (p=.592). Following three cycles of therapy, MMR was observed in 79.3% of the sorafenib group and 46.9% of the control group (p =.009). Sorafenib group also had a significantly higher CMR than control group (62.1% vs 28.1%, p =.009). After four cycles of therapy, MMR increased to 100% in the sorafenib group and 70.8% in the control group (p =.019), whileCMR was also more frequently achieved in the sorafenib group than that in the control group (90.9% vs 54.2%, p =.006). There was no significant difference of grades 3-4 adverse events between the two groups. Grade 4 neutropenia (neutrophil count less than 0.5×10⁹ cells per L) and thrombocytopenia (platelet count less than 20×10⁹ per L) were observed in all cases. The median duration of neutropenia was 12d (range, 6-25d) in the sorafenib group and 10d (range, 5-22d) in the control group (p =.063) , while the median duration of thrombocytopenia was 12d (range, 5-33d) and 9d (range, 6-19d) (p =.052), respectively.The most common grade 3-4 non-hematological adverse events in sorafenib and control group were pneumonia (31.3% vs 57.4%, p=.019), mucositis (21.9% vs 24.1%, p =.816), blood stream infection (37.5% vs 31.5%, p =.568). Conclusion Sorafenib combined with chemotherapy significantly increased the CMR after 4 cycles of therapy, however, the incidence of haematological and non-haematological adverse events did not apparently increased. The impact of sorafenib combined with chemotherapy on relapse and survival of CBF-AML need further following-up. Keywords: Sorafenib, CBF-AML, CMR, adverse events

Altered Cellular Sedimentation during Apheresis Impacts Outcomes of Peripheral Blood Stem Cell Collection Efficiency in Patients with Sickle Cell Disease

Recent reports have demonstrated the potential for gene therapy as a therapeutic option to achieve a durable cure in patients with sickle cell disease (SCD). However, for these emerging cell therapies, relatively large numbers of peripheral blood hematopoietic stem cells (PB HSCs) are required to generate a therapeutic dose. Outcomes of stem cell collections in patients with SCD are highly variable, at times yielding far fewer numbers of HSCs in the product than predicted by the number of PB CD34+ cells. While the CXCR4 antagonist plerixafor has emerged as a safe and reliable drug to mobilize CD34+ cells in patients with SCD, collection failures continue to occur requiring additional cycles or failure to generate a gene therapy product. We sought to understand the mechanics affecting stem cell sedimentation in real time and posited that SCD disease associated changes to red cells results in altered HSC sedimentation during apheresis, negatively impacting collection. Methods Autologous PB stem cell collections were performed in patients with SCD following mobilization with 240ug/kg plerixafor. To uncover the mechanisms impacting apheresis efficiency for collecting PB HSCs, we devised a method to interrogate the sedimentation of cells in peripheral blood in real time, by interrupting the return collection circuit line. Cellular composition analyses of the collecting interfaces were performed including CD34+ enumeration by flow cytometry, CBC and differential, reticulocyte counts, and surface CD47 expression of red blood cells (RBCs). Results Patients were mobilized with 240ug/kg of plerixafor up to 6 hours before apheresis collection. We collected fewer cells than predicted (3.78 million cells/kg) in these patients with an overall average apheresis collection efficiency of 38%, and a strikingly wide range of 2%-107%. We determined that the lower collection efficiency outcomes were primarily derived when using standard apheresis interface settings. The pre-apheresis PB CD34+ counts were less predictive of total CD34+ cells collected (p=0.012) in patients with SCD (r2=0.2557, p=0.0023) when compared to historical control patients without SCD (r2=0.7638, p<0.0001). Real time measurement of cell counts at standard (Hematocrit 3%, S) and deep (Hematocrit 5%, D) collecting interfaces of the apheresis machine showed a significant decrease in lymphocytes (p=0.0146), increase in monocytes (p=0.0496), and increase enrichment in CD34+ cells at the D interface (Panel A). Enrichment of CD34+ cells correlated with monocyte enrichment and (n=21, r2=0.6706, p<0.0001) and an increased fold change in CD34+ cells between the S and D interfaces correlated with higher PB reticulocyte percentage (r2=0.5295, p=0.0111). Further analysis of the individual collection interfaces revealed that most RBCs were HbS containing despite a PB HbS concentration <30%, consistent with an enrichment for shorter lived sickle red cells. CD47 expression has been previously reported in higher concentrations on the surface of younger RBCs during erythropoiesis. Surface expression of CD47 was found to be significantly higher at both the S and D interfaces compared to the PB of patients, indicating that the accumulation of a younger population of RBCs at the collecting interfaces. As microcytic RBCs have been associated with poor collection efficiencies, the SCD red cell hemoglobin concentration in the PB was used as a marker for size and a significant correlation was determined (r2=0.6086, p=0.0001). Patients with SCD can mobilize PB HSC in response to plerixafor but demonstrate specific characteristics that impede the collection of stem cells. During apheresis, CD34+ stem cells sediment at a deeper hematocrit interface in the buffy coat as a result of displacement by the younger patient-derived population of RBCs. The relative shift in erythrocyte sedimentation requires a corresponding alteration in collection strategies to improve the collection efficiency. In addition to red cell age, other factors that affect red cell buoyancy including iron status or additional globin gene mutations negatively impact apheresis stem cell collections. Full assessment of red cell parameters can identify patients that are at risk for poor stem cell collection outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.

Abstract P1121: Transposition Of Great Arteries - Genetics And Beyond

Background: Transposition of great arteries (TGA) is one of the most common severe congenital heart defects and neonatal cyanotic heart diseases. Given contribution of several molecular pathways involved in cardiac differentiation (Fig.1), the etiology governing morphogenesis of TGA remains largely unknown. There is greater insight into interactions and molecular variations culminating in aortic-pulmonary transposition. Clinical evidence and genetic sequencing has led to several polymorphic mutations demonstrating phylogenetic conservation of regulatory domains. Defects in transcription factors involved in nodal signaling, in addition to ZIC3 &amp; PROSIT 240, are strongly implicated in arterial transposition. We reviewed current literature on the genetics determining TGA development and associated lateral syndromes Methods: We searched PubMed, Scopus, Web of Science, and Google Scholar to identify listed keywords Body: TGA is now postulated as part of a spectrum of genetic disorders with familial recurrence, rather than sporadic defect. Evidence exists for TGA's correlation with genes involving lateralization defects, heterotaxy and asplenia. Evidence, albeit scarce, has surfaced depicting association of TGA with genetic syndromes like Trisomy 8, 18, VACTERL and CHARGE syndrome, Tuberous sclerosis, Deletion 11q, 18p, and other genetic syndromes like Digeorge, Turner’s, Noonan’s, Williams, and Marfan. Additional gene mutations causing TGA might include CFC-1, TRAP2, FOXH1, LEFTY2, PITX2, GDF1. The interrelation between mutations in ciliary genes contributing to both TGA and neurodevelopment disorder, prompts consideration of common ciliopathic pathogenies explaining association between TGA and neurodevelopmental disorders. Conclusion: The occurrence of mutations in a single gene, though a cause for TGA, does not correlate with 5-7% occurrence rate for TGA. Major research has focused on a single gene approach, neglecting the simultaneous screening for other susceptible targets. The multiple-hit model suggested by De Luca et al. (2010) is a promising approach for further understanding the contribution of multi-gene sequences. Minor genetic variations in several genes may have a cumulative effect that increases disease susceptibility in TGA and other congenital heart defects

Mutation in the Two-Component System Regulator YycH Leads to Daptomycin Tolerance in Methicillin-Resistant Staphylococcus aureus upon Evolution with a Population Bottleneck.

ABSTRACTAdaptive laboratory evolution (ALE) is a useful tool to study the evolution of antibiotic tolerance in bacterial populations under diverse environmental conditions. The role of population bottlenecks in the evolution of tolerance has been investigated in Escherichia coli, but not in a more clinically relevant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used ALE to evolve MRSA under repetitive daptomycin treatment and incorporated population bottlenecks following antibiotic exposure. We observed that the populations finally attained a tolerance mutation in the yycH gene after 2 weeks of evolution with population bottlenecks, and additional mutations in yycI and several other genes further increased the tolerance level. The tolerant populations also became resistant to another glycopeptide antibiotic, vancomycin. Through proteomics, we showed that yycH and yycI mutations led to the loss of function of the proteins and downregulated the WalKR two-component system and the downstream players, including the autolysin Atl and amidase Sle1, which are important for cell wall metabolism. Overall, our study offers new insights into the evolution of daptomycin tolerance under population bottlenecking conditions, which are commonly faced by pathogens during infection; the study also identified new mutations conferring daptomycin tolerance and revealed the proteome alterations in the evolved tolerant populations.IMPORTANCE Although population bottlenecks are known to influence the evolutionary dynamics of microbial populations, how such bottlenecks affect the evolution of tolerance to antibiotics in a clinically relevant methicillin-resistant S. aureus (MRSA) pathogen are still unclear. Here, we performed in vitro evolution of MRSA under cyclic daptomycin treatment and applied population bottlenecks following the treatment. We showed that under these experimental conditions, MRSA populations finally attained mutations in yycH, yycI, and several other genes that led to daptomycin tolerance. The discovered yycH and yycI mutations caused early termination of the genes and loss of function of the proteins, and they subsequently downregulated the expression of proteins controlled by the WalKR two-component system, such as Atl and Sle1. In addition, we compared our proteomics data with multiple studies on distinct daptomycin-tolerant MRSA mutants to identify proteins with a consistent expression pattern that could serve as biological markers for daptomycin tolerance in MRSA.

116 Identification of germline pathogenic mutations in patients with high-frequency basal cell carcinomas

Basal cell carcinoma (BCC) is the most common malignancy worldwide with more than 4 million new cases annually in the US. Though BCCs develop from UV radiation, host genetics also play a role, as demonstrated by patients with Basal Cell Nevus Syndrome (BCNS), who carry germline PTCH1 mutations. Genome-wide association studies have also identified common genetic polymorphisms associated with BCC. Interestingly, frequent BCC development has been associated with risk of solid tumor malignancy. This study investigates germline variants that contribute to development of high-frequency BCCs (hfBCCs) in patients without BCNS. We performed germline whole genome sequencing in 88 patients with hfBCCs, defined as ≥6 in a 10-year period (mean 14.9 BCCs, range 6-98). 27.7% of hfBCC patients reported an internal malignancy. On average 2.2 pathogenic or likely pathogenic mutations per patient (range 0-11) were identified by ClinVar or InterVar. Recurrent mutations were seen in DNA repair, tumor suppressor, immune regulation, and pigmentation genes. Highly penetrant mutations included an EPHB2 variant, associated with prostate and brain cancer, in 5.7% of our cohort (enriched by 10.7-fold in comparison to Exome Aggregation Consortium Non-Finnish European (ExAC NFE) population frequency). In addition, 3.4% of patients had mutations in PADI3 (2.2-fold increase compared to ExAC NFE), associated with uncombable hair syndrome. Notably, 80% of patients with EPHB2 variant and 67% of patients with PADI3 mutations had a history of internal malignancy. Additional gene mutations identified include TYR, PRKN, MITF, EYS, JAK2, MLH3, and CHEK2. Our findings identify germline pathogenic mutations, some present in several patients with internal or hematopoietic cancers. We are currently exploring the functional implications of these genes in cell line assays to elucidate how they play a role in cancer susceptibility and development.

CD4 bovine gene: Differential polymorphisms among cattle breeds and a new tool for rapid identification

Two mutations in the CD4 bovine gene (G>T/Q306H; A>C/K310N) were identified as causative for altered staining with anti-CD4 mAb #CC8. We developed a HRM qPCR for genotyping these mutations and compare with immunophenotyping in different cattle breeds. The assay distinguished five genotypes, B (homozygous, G/A) and C (heterozygous, G/A and T/C), found in taurine, A (homozygous, G/C) and D (heterozygous, T/C and G/C), found in zebu. The E genotype (homozygous, T/C) was not observed in tested animals. As expected, B and C presented high/very high and intermediate CD4 staining, respectively. The lack/low CD4 staining was mainly related to the A, while the intermediate staining was mainly related to D genotype. The developed HRM qPCR assay accurately identified the altered phenotypes associated with CC8 staining in taurine. However, the assay cannot be applicable in zebu or hybrid breeds, probably due to additional mutations in the CD4 gene from zebu descendant animals.

Evidence of neural crest cell origin of a DICER1 mutant CNS sarcoma in a child with DICER1 syndrome and NRAS-mutant neurocutaneous melanosis.

Evidence of neural crest cell origin of a DICER1 mutant CNS sarcoma in a child with DICER1 syndrome and NRAS-mutant neurocutaneous melanosis.

P670: ABSENCE OF BTK, BCL2, AND PLCG2 MUTATIONS IN RELAPSING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AFTER FIRST-LINE TREATMENT WITH FIXED-DURATION IBRUTINIB (I) PLUS VENETOCLAX (V)

Background: CAPTIVATE (PCYC-1142; NCT02910583) is a multicenter phase 2 study of first-line I+V in patients (pts) with CLL in 2 cohorts: Minimal Residual Disease (MRD) and Fixed Duration (FD). We previously reported that fixed-duration I+V provides deep, durable responses (Ghia et al, ASCO 2021; Wierda et al, J Clin Oncol 2021). Clinically-relevant mutations in BTK, PLCG2, and BCL2 have previously been reported in pts with progressive disease (PD) following single agent BTK or BCL2 inhibitor therapies. Such resistance-associated mutations may occur with increasing frequency after prolonged drug exposure. Fixed duration I+V is hypothesized to decrease the likelihood of developing resistance mutations and facilitate extended clinical benefit. Aims: We sought to ascertain presence or absence of resistance mutations at the time of PD in samples from pts treated with fixed-duration I+V in CAPTIVATE. Baseline genomic risk features and potentially prognostic mutations in CLL-related genes of interest were also evaluated in pts with and without subsequent PD. Methods: The analysis was conducted in 191 pts aged ≤70 y with previously untreated CLL who completed fixed-duration I+V, defined as 3 x 28d cycles of I then 12 cycles of I+V (I 420 mg/d orally; V ramp-up to 400 mg/d orally); this included pts from the FD cohort, and pts from the MRD cohort with confirmed undetectable MRD who received an additional cycle of I+V and were randomized to placebo. Samples from pts at the time of PD were evaluated for resistance mutations by targeted next-generation sequencing (NGS) using a custom-designed high-read-depth 100-gene panel (limit of detection 1% VAF, negative predictive value ≥95% assuming population prevalences <20%, for BTK, PLCG2, or BCL2 variants) in CD19-enriched peripheral blood (PB) or bone marrow. Mutations in additional genes of interest (Table) were evaluated by NGS using the Personalis ACE panel in baseline PB samples. Genomic risk features (Table) were also assessed at baseline. Mutations and genomic risk features evident at baseline were compared across PD and non-PD pts. Results: Baseline mutations in genes of interest and genomic risk features in 190 pts who completed fixed-duration I+V and with available baseline data are shown in the Table. Rates of mutations were similar between pts with (n=30) and without (n=160) subsequent PD. Of 30 pts with PD events (occurring between 16 and 53 mos after start of treatment), 22 (73%) had baseline mutations in genes of interest and 27 (90%) had ≥1 baseline genomic risk feature. Pts with PD generally had more baseline genomic risk features than pts without PD (median 3 vs 2; Wilcoxon P=0.02; analysis restricted to FD cohort pts to avoid bias). To date, 26 of 30 PD patient samples have been evaluated for mutations; no resistance-associated variants in BTK, PLCG2, or BCL2 were detected. Image:Summary/Conclusion: Earlier work has demonstrated a promising safety profile and efficacy of fixed-duration I+V in first-line CLL. Importantly, the current results show that no resistance-associated mutations in BTK, PLCG2, or BCL2 were identified in the pts with PD evaluated to date after first-line, fixed-duration I+V. The absence of such mutations at PD suggests that fixed-duration I+V treatment may offer the possibility of effective subsequent retreatment options with I and/or V and extend clinical benefit.

Abstract LB113: Genomic classification to refine prognosis in clear cell renal cell carcinoma

Abstract Renal cell carcinomas (RCC) are characterized by their heterogenous clinical outcomes, and due to their indeterminate behavior and the absence of routine biomarkers, it is difficult to identify patients who are at high-risk for relapse after curative nephrectomy. To identify genomic biomarkers for clear cell RCC (ccRCC) risk-stratification we interrogated somatic mutation status of 12 RCC-relevant genes using next-generation sequencing (NGS) in tumor-normal pairs from 943 patients with matched follow up data from the Cancer Genomics of the Kidney (CAGEKID) study. We examined associations between genomically-defined patient groups, explained below, and disease-free as well as RCC-specific survival independently in two cohorts of patients (N=469 for cohort 1; 474 for cohort 2). We used the Kaplan-Meier method with log-rank tests to compare survival functions, and Cox proportional hazards models to stratify for patient stage and age to estimate association of each group with survival. RCC-specific survival was assessed with a competing-risks method to include deaths from other causes. Within these cohorts, 76.4% of patients harbored somatic mutations in VHL, the most common driver gene in ccRCC. The most commonly mutated genes within VHL-mutated tumors were PBRM1 (39.7%), SETD2 (19%), BAP1 (14.3%), and KDM5C (8.3%). Less frequently mutated genes included ATM, COL11A1, DMD, TP53, and TRRAP (~3-5%).Among VHL-driven tumors, we identified a new genomic classifier on the basis of the number of mutations in additional RCC driver genes in the panel examined. Patients were classified based on the presence of mutations only in VHL (VHL+0), those with mutations in VHL and one other driver gene (VHL+1), two other driver genes (VHL+2), and 3 or more other driver genes (VHL≥3). We observed within both cohorts that both the risk of disease recurrence as well as RCC-specific death were associated with an increased number of mutations within this classification. When stratified for patient stage and age, the hazard-ratio for 5-year disease-free survival for VHL≥3 patients was 6.69 (p=0.000212), 4.31 for VHL+2 (p=0.000862), and 2.43 for VHL+1 (p=0.035662), compared to patients with only mutations in VHL. These observations were replicated in the second patient cohort, with hazards ratios of 4.55, 2.49, and 1.40, for VHL≥3, VHL+2, and VHL+1 classified patients respectively, indicating that risk of disease recurrence increases with the number of driver mutations. Notably, tumor mutational burden (TMB) was not significantly different between the aforementioned groups, demonstrating that our classifier is independent of TMB. We created a model based on a set of 12 RCC-relevant genes, which can predict risk of relapse for the ~80% of patients with ccRCC that are VHL-driven. This classification can be defined based on a small panel of genes, making it easily applicable to the clinic, in the context of tumor or liquid biopsy analysis. Citation Format: Kate I. Glennon, Naveen S. Vasudev, Ghislaine Scelo, Michelle Wilson, Louis Letourneau, Robert Eveleigh, Nazanin Nourbehesht, Madeleine Arseneault, Antoine Paccard, Lars Egevad, Juris Viksna, Edgars Celms, Sharon M. Jackson, Behnoush Abedi-Ardekani, Anne Y. Warren, Peter J. Selby, Sebastian Trainor, Michael Kimuli, Naeem Soomro, Adebanji Adeyoju, Poulam Patel, Magdalena B. Wozniak, Ivana Holcatova, Antonin Brisuda, Vladimir Janout, Estelle Chanudet, David Zaridze, Anush Moukeria, Oxana Shangina, Lenka Foretova, Marie Navratilova, Dana Mates, Viorel Jinga, Ljiljana Bogdanovic, Bozidar Kovacevic, Anne Cambon-Thomsen, Guillaume Bourque, Alvis Brazma, Jörg Tost, Paul Brennan, Mark Lathrop, Yasser Riazalhosseini, Rosamonde E. Banks. Genomic classification to refine prognosis in clear cell renal cell carcinoma [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 LB113.

Abstract 3323: Discovery of a small-molecule inhibitor that can target EGFR with C797S mutation

Abstract EGFR is a transmembrane protein that functions as a receptor tyrosine kinase (RTK). Upon ligand binding or by activating mutations (Exon19 deletion, L858R mutation, and others), EGFR turns on the downstream signals that include oncogenic RAS/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT pathways. EGFR gene mutations and amplifications are frequently found in various human cancers and, in non-small cell lung cancer (NSCLC), the EGFR gene is mutated with 10-15% frequency (about 50% in Asian patients). While the 1st and 2nd generation EGFR inhibitors are effective in targeting EGFR mutants with Exon19 deletion and L858R mutation, additional T790M mutation in the EGFR gene causes resistance. The 3rd generation EGFR inhibitor (Osimertinib) works against EGFR mutants with T790M mutation; however, another EGFR mutation occurs at the amino acid 797 position (C797S), which makes Osimertinib ineffective. This is one of the major resistance mechanisms in Osimertinib-treated patients, and the 4th generation EGFR inhibitor that can target the C797S mutant is needed. We developed a compound that effectively disables various EGFR mutations, including Del19/T790M/C797S, L858R/T790/C797S, Del19/C797S, and L858R/C797S. In vitro kinase assay and cellular assays showed high potency and selectivity. In vivo PK/PD and efficacy tests confirmed the great therapeutic potential of this inhibitor for patients with EGFR mutations. Citation Format: Dongsu Kim, Woo Seung Son, Anna Jang, Yeri Lee, Donggeon Kim, Changyu Choi, Kyung Hoon Min, Sung Pil Choi, Sang Kyun Lim. Discovery of a small-molecule inhibitor that can target EGFR with C797S mutation [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 3323.

Abstract 1615: Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells

Abstract The genetic tumor predisposition syndrome Neurofibromatosis type 1 (NF1) results from the inheritance of a mutant copy of NF1, a RAS-GAP tumor suppressor gene. Subsequent loss of the remaining wild-type NF1 allele in Schwann lineage cells of the peripheral nervous system leads to complete functional loss of the encoded protein, neurofibromin. Schwann cells lacking neurofibromin exhibit hyperactive RAS signaling, and contribute to the formation of benign plexiform neurofibromas (PNFs). Through additional mutations in tumor suppressor genes CDKN2A/CDKN2B, these PNFs may escape senescence and progress to atypical neurofibromatous neoplasms of uncertain biological potential (ANNUBP). Furthermore, loss of function mutations in Polycomb Repressive Complex 2 (PRC2) have been strongly implicated in the progression of ANNUBPs to lethal malignant peripheral nerve sheath tumors (MPNSTs). Although these alterations have been associated with MPNST formation, their temporal dependence during Schwann cell development and contribution to malignant transformation using a human cell model has not been studied. We hypothesize that sequential loss of NF1, CDKN2A, and finally SUZ12 in Schwann cells transforms them into MPNST, and this process is possible to study using a human induced pluripotent stem cell (iPSC) model system. To test our hypothesis, we generated Schwann lineage cells from commercially available iPSCs. Cells were subjected to targeted mutations using CRISPR/Cas9 ribonucleoproteins at different stages of development to mimic potential tumorigenesis. Our preliminary results show that multiple iPSC donor lines can reliably be differentiated to Neural Crest Cells (NCCs), and further to mature Schwann cells. We have edited multiple target loci at high levels (&amp;gt;70%) throughout Schwann cell development. NCCs lacking neurofibromin showed an increase in proliferation and migration. While iPSC-derived Schwann cells (iSCs) lacking NF1 and CDKN2A with or without SUZ12 loss became senescent after several passages. These mutant iSCs were unable to form tumors when subcutaneously injected into the flank of NRG mice, while commonly utilized immortalized human Schwann cell lines lacking NF1 were able to form large subcutaneous tumors. Further refinement of this induced-MPNST (iMPNST) model through targeting of CDKN2B, ATRX, or TP53 may allow for better understanding of the order and timing at which these transforming mutations are sufficient to result in MPNST formation. Additionally, the application of single-cell RNA sequencing technology to this system could uncover potential heterogeneity that could harbor a clone primed for malignant transformation. This model could aid in uncovering novel genetic mutations and pathways which contribute to transformation of benign PNFs to MPNSTs, providing additional targets for future therapeutic intervention. Citation Format: Garrett M. Draper, Daniel Panken, Mahathi Patchava, Wendy Hudson, Kyle B. Williams, David A. Largaespada. Developing a novel model of neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors using induced pluripotent stem cells [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 1615.

Are human induced pluripotent stem cell derived cardiomyocytes a good cellular model for studying incomplete penetrance in arrhythmogenic cardiomyopathy?

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Regional Development Fund and Interreg V-A Italy-Austria 2014-2020 and Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy). Background Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were previously used to model several inherited heart diseases (1). Among these, arrhythmogenic cardiomyopathy (ACM) is characterized by a fibrofatty myocardial replacement and severe ventricular arrhythmias (2). ACM is mainly caused by mutations affecting proteins of intercalated discs (3) and shows incomplete penetrance and variable expressivity (4). Purpose To evaluate whether hiPSC-CMs provide a good in vitro model to study incomplete penetrance in ACM. Material and methods hiPSCs were generated from buffy coats of 6 close relatives. Three of them were ACM patients carrying a deletion of exon 4 in the PKP2 gene causing a premature stop codon, 2 were asymptomatic (ASY) PKP2 mutation carriers and 1 was a healthy control (CTR). Whole exome sequencing of the 6 individuals was performed. The purified hiPSC-CMs were cultured in basal and adipogenic medium and examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp and immunofluorescence. Results All family members tested negative for additional mutations in ACM genes. As expected, half the amount of wild type (wt) PKP2 mRNA was found in ACM and ASY than in CTR hiPSC-CMs (2.25±0.27vs4.29±0.70 wtPKP2/HPRT1; p=0.028) (2.08±0.38vs4.29±0.70 wtPKP2/HPRT1; p=0.052). Of note, the mutated (mut) PKP2 mRNA was detected and significantly more expressed in ACM than in ASY hiPSC-CMs (0.33±0.04vs0.11±0.06 mutPKP2/HPRT1; p=0.029). While we confirmed a reduced amount of wt plakophillin-2 protein in ACM and ASY compared to CTR hiPSC-CMs (0.19±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.016), (0.25±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.11), the truncated protein was not detected. Moreover, a significantly lower expression of the active form of β-catenin (ABC) has been shown in ACM and ASY than in CTR hiPSC-CMs (0.93±0.18vs1.30±0.09 ABC/GAPDH; p=0.057), (0.76±0.10vs1.30±0.09 ABC/GAPDH; p=0.0057). In basal medium, ACM hiPSC-CMs had a significant lower overall sodium current density compared to CTR (-49.82±2.8 vs -69.38±4.8 pA/pF; p&amp;lt;0.0001) and to ASY (-49.82±2.8 vs -68.92±4.3 pA/pF; p&amp;lt;0.0001) hiPSC-CMs. In adipogenic medium, ACM hiPSC-CMs showed a higher lipid accumulation and a higher sarcomere disorganization than CTR (1580±219vs615±199 intensity/nuclei; p=0.0351) (0.019±0.001vs0.028±0.003 myofibril alignment index; p=0.039) and ASY (1580±219vs689±146 intensity/nuclei; p=0.0059) (0.019±0.001vs0.027±0.001 myofibril alignment index; p=0.010) hiPSC-CMs. Conclusions Here we report that ACM express a significantly higher amount of mutated PKP2 mRNA than ASY iPSC-CMs. Even though no differences were detected for plakophilin-2 and active β-catenin proteins between ACM and ASY iPSCM-CMs, the ACM showed a fatty phenotype and altered electrical activity that differed significantly from the CTR as well as from the ASY hiPSC-CMs, demonstrating that these cells provide a valid model to study incomplete penetrance in ACM.

A Systematic Review of the Role of Runt-Related Transcription Factor 1 (RUNX1) in the Pathogenesis of Hematological Malignancies in Patients With Inherited Bone Marrow Failure Syndromes.

Somatic runt-related transcription factor 1 (RUNX1) mutations are the most common mutations in various hematological malignancies, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Mono-allelic RUNX1 mutations in germline cells may cause familial platelet disorder (FPD), an inherited bone marrow failure syndrome (IBMFS) associated with an increased lifetime risk of AML. It is suspected that additional RUNX1 mutations may play a role in the pathogenesis of hematological malignancies in IBMFS. This review aims to study the role of RUNX1 mutations in the pathogenesis of hematological malignancies in patients with IBMFS. A PubMed database search was conducted using the following medical subject heading (MeSH) terms: “inherited bone marrow failure syndromes,” “hematological neoplasms,” “gene expression regulation, leukemic,” “RUNX1 protein, human,” “RUNX1 protein, mouse,” and “Neutropenia, Severe Congenital, Autosomal recessive.” Three studies published in 2020 were identified as meeting our inclusion and exclusion criteria. Leukemic progression in severe congenital neutropenia was used as a disease model to evaluate the clinical, molecular, and mechanistic basis of RUNX1 mutations identified in hematological malignancies. Studies in mice and genetically reprogrammed or induced pluripotent stem cells (iPSCs) have shown that isolated RUNX1 mutations are weakly leukemogenic and only initiate hyperproduction of immature hematopoietic cells when in combination with granulocyte colony-stimulating factor 3 receptor (GCSF3R) mutations. Despite this, whole-exome sequencing (WES) performed on leukemogenic transformed cells revealed that all AML cells had an additional mutation in the CXXC finger protein 4 (CXXC4) gene that caused hyperproduction of the ten-eleven translocation (TET2) protein. This protein causes inflammation in cells with RUNX1 mutations. This process is thought to be critical for clonal myeloid malignant transformation (CMMT) of leukemogenic cells. In conclusion, the combinations of GCSF3R and RUNX1 mutations have a prominent effect on myeloid differentiation resulting in the hyperproduction of myeloblasts. In other studies, it has been noted that the mutations in GCSF3R and RUNX1 genes are not sufficient for the full transformation of leukemogenic cells to AML, and an additional clonal mutation in the CXXC4 gene is essential for full transformation to occur. These data have implicitly demonstrated that RUNX1 mutations are critical in the pathogenesis of various hematological malignancies, and further investigations into the role of RUNX1 are paramount for the development of new cancer treatments.

Comprehensive Analysis of Acquired Genetic Variants and Their Prognostic Impact in Systemic Mastocytosis.

Simple SummarySystemic mastocytosis (SM) is a clonal haematopoietic stem cell disease typically characterized by the expansion and accumulation of neoplastic mast cells carrying the activating KIT D816V as a driver mutation. Multilineage involvement of haematopoiesis by this KIT mutation, particularly in a multi-mutated context, also involving other genes (e.g., SRSF2, ASXL1, DNMT3A, RUNX1, EZH2, CBL and NRAS) found to be frequently mutated in other myeloid neoplasms, have recently emerged as a genetic background associated with malignant transformation of SM. Therefore, assessment of multilineage involvement of haematopoiesis by KIT D816V and additional mutations in genes known to be associated with the prognosis of SM have become of great help to identify good vs. poor-prognosis SM patients who could benefit from a closer follow-up and, eventually, also early cytoreductive treatment.Systemic mastocytosis (SM) is a rare clonal haematopoietic stem cell disease in which activating KIT mutations (most commonly KIT D816V) are present in virtually every (>90%) adult patient at similar frequencies among non-advanced and advanced forms of SM. The KIT D816V mutation is considered the most common pathogenic driver of SM. Acquisition of this mutation early during haematopoiesis may cause multilineage involvement of haematopoiesis by KIT D816V, which has been associated with higher tumour burden and additional mutations in other genes, leading to an increased rate of transformation to advanced SM. Thus, among other mutations, alterations in around 30 genes that are also frequently mutated in other myeloid neoplasms have been reported in SM cases. From these genes, 12 (i.e., ASXL1, CBL, DNMT3A, EZH2, JAK2, KRAS, NRAS, SF3B1, RUNX1, SF3B1, SRSF2, TET2) have been recurrently reported to be mutated in SM. Because of all the above, assessment of multilineage involvement of haematopoiesis by the KIT D816V mutation, in the setting of multi-mutated haematopoiesis as revealed by a limited panel of genes (i.e., ASXL1, CBL, DNMT3A, EZH2, NRAS, RUNX1 and SRSF2) and associated with a poorer patient outcome, has become of great help to identify SM patients at higher risk of disease progression and/or poor survival who could benefit from closer follow-up and eventually also early cytoreductive treatment.

Treatment pattern and outcomes in de novo T790M-mutated non-small cell lung cancer

IntroductionLimited data exists for non-small cell lung cancer (NSCLC) patients harbouring de novo T790M mutation.MethodsNSCLC patients, with de novo T790M, who registered at our institute between 01/03/2015 and 31/12/2019, were considered for retrospective analysis of treatment pattern and clinical outcomes, i.e., progression-free survival (PFS) and overall survival (OS).ResultsOf 1,542 epidermal growth factor receptor (EGFR)-mutated patients, 40 (2.59%) had de novo T790M. Most were male (27, 67.5%) and smokers (23, 57.5%). The commonest site of metastasis was the lungs (31, 77.5%), while 7 (17.5%) had central nervous system (CNS) involvement. Additional EGFR gene mutations and anaplastic lymphoma kinase (ALK) positivity were observed in 20 (50.0%) and 4 (10.0%) cases, respectively. The first-line systemic therapy and the number of patients receiving it were as follows: osimertinib by 14 (35.0%), first-generation EGFR tyrosine kinase inhibitors (TKIs) by 10 (25.0%), gefitinib + chemotherapy by 3 (7.5%), chemotherapy by 7 (17.5%) and gefitinib + bevacizumab by 2 (5%). One patient defaulted before starting any treatment. Hence, 39 were considered for survival analysis. The median PFS and OS for the entire cohort were 10.4 (95% CI = 7.6–19.7) months and 24.9 (95% CI = 15.7–NA) months, respectively. The median PFS for patients on osimertinib was 19.8 (95% CI = 11.6–28.0) months versus 8.8 (95% CI = 6.6–10.9) months for those on other systemic therapy. No CNS involvement, use of osimertinib or first-generation EGFR TKI plus chemotherapy or ALK inhibitor in ALK-positive cases prognosticated better PFS. When compared to other systemic therapies, osimertinib improved PFS in patients with or without additional EGFR mutations, although it was statistically significant for the former group only (p = 0.002).ConclusionThe incidence of de novo T790M is low. Osimertinib in frontline therapy provides promising outcomes.

Differential VHL Mutation Patterns in Bilateral Clear Cell RCC Distinguishes Between Independent Primary Tumors and Contralateral Metastatic Disease

ObjectiveTo evaluate whether bilateral, multifocal clear cell renal cell carcinoma (ccRCC) patients can be differentiated by VHL mutation analysis into cases that represent either multiple independently arising primary tumors, or a single primary tumor which has spread ipsilaterally as well as to the contralateral kidney. The nature of kidney cancer multifocality outside of known hereditary syndromes is as yet poorly understood. Materials and MethodsDNA from multiple tumors per patient were evaluated for somatic VHL gene mutation and hypermethylation. A subset of tumors with shared VHL mutations were analyzed with targeted, next-generation sequencing assays. ResultsThis cohort contained 5 patients with multiple tumors that demonstrated a shared somatic VHL mutation consistent with metastatic spread including to the contralateral kidney. In several cases this was substantiated by additional shared somatic mutations in ccRCC-associated genes. In contrast, the remaining 14 patients with multiple tumors demonstrated unique, unshared VHL alterations in every analyzed tumor, consistent with independently arising kidney tumors. None of these latter patients showed any evidence of local spread or distant metastasis. ConclusionThe spectrum of VHL alterations within evaluated bilateral, multifocal ccRCC tumors from a single patient can distinguish between multiple independent tumor growth and metastasis. This can be performed using currently available clinical genetic tests and will improve the accuracy of patient diagnosis and prognosis, as well as informing appropriate management.

Clinical impact and proposed application of molecular markers, genetic variants, and cytogenetic analysis in mast cell neoplasms: Status 2022

Mast cell neoplasms are an emerging challenge in the fields of internal medicine, allergy, immunology, dermatology, laboratory medicine, and pathology. In this review, we discuss the current standards for the diagnosis and prognostication of mast cell neoplasms with special reference to clinically relevant germline and somatic gene variants. In patients with cutaneous mastocytosis or with indolent systemic mastocytosis (SM), various KIT-activating mutations act as key molecular drivers of the disease. In adults, KIT p.D816V is by far the most prevalent driver, whereas other KIT mutants are detected in nearly 40% of children. In advanced SM, including aggressive SM, SM with an associated hematological neoplasm, and mast cell leukemia, additional somatic mutations in other genes, such as SRSF2, JAK2, RUNX1, ASXL1, or RAS, may be detected. These drivers are more frequently detected in SM with an associated hematological neoplasm, particularly in male patients. Recently, hereditary alpha-tryptasemia has been identified as a genetic trait more prevalent in SM compared with healthy controls. Moreover, hereditary alpha-tryptasemia is more frequent in patients with SM with Hymenoptera venom allergy and severe mediator-related symptoms than in patients with SM without symptoms. On the basis of this knowledge, we propose a diagnostic algorithm in which genetic markers are applied together with clinical and histopathologic criteria to establish the diagnosis and prognosis in SM.

TMPRSS2-ERG promotes the initiation of prostate cancer by suppressing oncogene-induced senescence.

ERG translocations are commonly involved in the initiation of prostate neoplasia, yet previous experimental approaches have not addressed mechanisms of oncogenic inception. Here, in a genetically-engineered mouse model, combining TMPRSS2-driven ERG with KrasG12D led to invasive prostate adenocarcinomas, while ERG or KrasG12D alone were non-oncogenic. In primary prostate luminal epithelial cells, following inducible oncogenic Kras expression or Pten depletion, TMPRSS2-ERG suppressed oncogene-induced senescence, independent of TP53 induction and RB1 inhibition. Oncogenic KRAS and TMPRSS2-ERG synergized to promote tumorigenesis and metastasis of primary luminal cells. The presence of TMPRSS2-ERG compared to a wild-type background was associated with a stemness phenotype and with relatively increased RAS-induced differential gene expression for MYC and mTOR-regulated pathways, including protein translation and lipogenesis. In addition, mTOR inhibitors abrogated ERG-dependent senescence resistance. These studies reveal a previously unappreciated function whereby ERG expression primes preneoplastic cells for the accumulation of additional gene mutations by suppression of oncogene-induced senescence.

Analysis of Coexisting Gene with NRAS in Acute Myeloid Leukemia

To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients. High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated. A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons. The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.