Non-canonical Mutation Research Articles

Germline MPL Mutations May be a Rare Cause of “Triple-Negative” Thrombocytosis

Background Rare cases of primary thrombocytosis are related to inherited genetic abnormalities and are called hereditary thrombocytosis (HT). HT has clinical features resembling sporadic essential thrombocythemia (ET) but has usually an autosomal dominant (AD) inheritance with variable penetrance. To date, germline mutation involving the MPL, THPO, JAK2 genes have been associated with HT. Methods The study included 933 patients evaluated at our Division because of persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Patients were screened for the three canonical somatic driver mutations associated with myeloproliferative neoplasms: JAK2 V617F mutation, CALR exon 9 mutations, MPL exon 10 mutations. Triple-negative cases with unexplained isolated thrombocytosis which also resulted negative for non-canonical exon 10 MPL mutations were analyzed using next generation sequencing (NGS) to test a panel of 80 myeloid genes. Results Of 933 patients with isolated thrombocytosis screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated and 68 triple-negative. Two MPL-mutated patients carried a non-canonical mutation located in exon 10: MPL W515* and MPL V501A. Of the 68 triple-negative patients, 32 were evaluated by NGS and one of them showed another non-canonical mutation of MPL located outside exon 10: MPL R102P. These 3 non-canonical MPL mutations resulted to be germline as they were detected also in DNA extracted from T lymphocytes and from hair roots. Due to the germline nature of the mutations, a study of the family was carried out. In 2 out of 3 pedigrees at least a second relative carried the same germline mutation discovered in the proband; these 2 pedigrees are shown in the figure. Patient MPC17_15 was an 80 years-old patient with a history of thrombocytosis. She resulted JAK2- and CALR- wild-type while HRM screening was positive for exon 10 MPL mutations; Sanger sequencing identified the MPL W515* mutation in DNA from granulocytes, T-lymphocytes and hair roots, confirming its germline nature. Her children showed a normal complete blood count (CBC) and were MPL wild-type, thus confirming a segregation between mutation and clinical phenotype. Patient MPC14_674 (panel A) was a 36 years-old women with a history of mild thrombocytosis never investigated before. She was referred to our clinic by Pediatricians after the delivery because her baby (MPC17_167) showed persistent isolated thrombocytosis (826 x10 9/L) during the first months of life despite the exclusion of the most common reactive causes of thrombocytosis. Sanger sequencing identified the MPL V501A mutation in granulocyte and in T-lymphocyte in both cases. To evaluate the segregation of MPL V501A with the disease phenotype, we evaluated the CBC and MPL mutation in the other relatives. Both proband's father (MPC23_597) and sister (MPC23_602) carried germline heterozygous MPL V501A mutation associated with a PLT count above or at the upper limit of normal range (570 x10 9/L and 355 x10 9/L, respectively). Conversely, the husband (MPC17_495), the second son (MPC18_43) and the mother (MPC23_603) of the proband showed a normal CBC and were MPL wild-type, confirming an AD inheritance. Patient MPC07_130 (panel B) was a 44 years-old women with triple-negative isolated thrombocytosis lasting since 2007. NGS analysis showed the presence of a heterozygous germline MPL R102P mutation. As this mutation is located in exon 3, it was not detected through HRM and Sanger sequencing which target only exon 10. Her son (MPC13_672), 18 years-old, was completely asymptomatic but the CBC showed mild thrombocytosis (472 x 10 9/L). He tested negative for the 3 driver mutations but Sanger sequencing of MPL exon 3 identified the heterozygous germline MPL R102P mutation. Conclusions We identified 3 non-canonical germline MPL mutations in 3 different unrelated subjects with isolated thrombocytosis: MPL W515*, MPL R102P, and MPL V501A. Germline MPL mutations may underly HT and should be evaluated by sequencing of the whole exon 10 of MPL gene or by NGS in cases with isolated thrombocytosis negative for the three canonical somatic driver mutations. Namely, patients with HT might present as sporadic cases, and in the initial workup they might be inappropriately diagnosed with triple-negative ET. A correct diagnosis is pivotal to avoid unnecessary treatments.

T Cell/Natural Killer (NK) Cell Transcriptional Profiles Are Associated with Response to Gilteritinib in FLT3-Mutated Acute Myeloid Leukemia

S.R. & A.C. equally contributed Background: Despite significative improvements in the management of FLT3-mutated AML and its prognosis since FLT3 inhibitors ( FLT3i) adoption, a significant proportion of patients results refractory or ultimately relapses after this targeted treatment. Gilteritinib is an oral and selective FLT3i with single-agent activity as salvage therapy in Relapsed/Refractory patients (R/R). The investigation of resistance mechanism to Gilteritinib is crucial to guide next therapeutic choices and improve drug effectiveness. Thus far, correlative studies have elucidated mainly intrinsic and mutational resistance mechanisms. However, less is known regarding potential extrinsic mechanisms and about the role of the immune system in influencing this FLT3i activity. Herein, we aimed at investigating intrinsic as well as novel extrinsic (immunological) mechanisms of resistance to Gilteritinib in routine clinical practice. Methods: A cohort of FLT3-mut AML patients treated with Gilteritinib at Hematology Institute of Bologna was selected. DNA/RNA were extracted from bone marrow (BM) samples pre-therapy and at first response assessment and screened by a custom NGS panel covering the full-coding sequence of 28 genes and a NanoString gene-expression analysis using the PanCancer IO 360 Panel. Data were collected in accordance with GCP and Helsinki declaration. NanoString data were acquired and analyzed using the nCounter FLEX Analysis System and the nSolver advanced analysis module (version 2.0.115). DESeq2 multi-factor design was employed to account for potential confounders. A public sc-RNA-seq dataset of newly diagnosed AMLs (Dufva et al. Cancer Cell 2020) was used to map DEGs between Responders and Non-Responders to Gilteritinib onto reference AML BM samples. Single-cell gene expression scores were computed using the UCell package in R. Results: Overall, 20 FLT3-mutated R/R AML patients receiving Gilteritinib as single-agent therapy were enrolled in this study. The median age at Gilteritinib therapy was 62 (range 33 - 77) years and 13/20 (65%) patients were male. Seventeen out of 20 (85 %) patients harbored a FLT3-ITD mutation, 2/20 (10 %) a TKD mutation and 1 (5 %) patient both mutations. Eleven out of 20 (55 %) patients had a concomitant NPM1 mutation. Patients were dichotomized into Responders (R) (n=9) and Non-Responders (NR) (n = 11) according to BM response. Intrinsic mechanisms of resistance were evaluated by NGS analyses on 13 patients (R=7 and NR=6). Firstly, FLT3 full-coding sequencing did not detect any clearly pathogenetic Non-Canonical (NC) mutation: M737I, M837I, P890L and P534L were four detected NC variants with a possible role in FLT3i sensitivity, however their low VAF (1-2 %) suggests a “passenger” role in this cohort. Secondly, evaluating variations in VAF of non- FLT3 variants detected pre- and post-therapy we observed a new variant onset in 4/6 (66.7 %) Gilteritinib NR patients, affecting genes involved in RAS pathway in all cases (1 NRAS, 2 KRAS and 1 PTPN11). Significant differences in immune transcriptional profiles were observed when comparing R and NR patients (R = 9 and NR = 11), including higher expression of genes reflecting T/NK function ( CD8A, GZMK, KLRK1, GNLY) and, more specifically, T-cell activity ( PRF1, EOMES, ZAP70, TNFSF8) in responders. Conversely, oncogenes involved in MAPK/Akt ( PROM1, ANGPT1, ANGPT2) or cell-cycle regulators ( CDKN2A, CCNA1) were overexpressed in NR patients (Fig. 1a). We next selected the top 30 DEGs between R and NR and we averaged gene expression across all BM samples. Gene set enrichment analysis (GSEA) revealed that approximately 25% of the significantly enriched pathways when using the top 30 DEGs as input were related to T-cell function / TCR signaling. By projecting the top 30 DEGs genes onto a sc-RNA-seq map of the AML tumor microenvironment, we found that they were largely expressed by CD8 + T EM, CD8 + T CM and NK cells (Fig. 1b). Summary/Conclusion: This study confirmed evolutionary trajectories often involving RAS pathway mutations as an intrinsic mechanism of resistance to Gilteritinib therapy. Of note, differences in immune transcriptomic profiles, involving mainly CD8+ T-Cells and NK-Cells, were associated with response, pointing to a previously unappreciated role of the immune system in mediating response to Gilteritinib.

Genome-wide maps of UVA and UVB mutagenesis in yeast reveal distinct causative lesions and mutational strand asymmetries.

Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine-adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair-deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma.

PATH-21. INVESTIGATING CHANGES IN IDH MUTATION STATUS AT GLIOMA RECURRENCE

Abstract PURPOSE Isocitrate dehydrogenase (IDH) mutation status is a key diagnostic and prognostic feature of gliomas. It is thought to occur early in glioma tumorigenesis and remains stable over time. We investigated our tumour bank to identify patients where IDH mutation status changed after glioma recurrence. METHODS We identified four patients with immunohistochemistry-recorded IDH mutation statuses that conflicted between multiple tumour samples taken over time. Tissue samples from the tumour bank were analyzed using methylation profiling and Sanger sequencing of IDH1 and IDH2 to verify IDH status. RESULTS In all four instances, we found that IDH mutation status in fact remained stable, and there were reasonable explanations as to why mutation status was misinterpreted. One tumour had a non-canonical IDH2 R172K mutation that is not routinely tested for. Two patients had perilesional samples, revealed by methylation profiling to contain a mixture of tumour and normal brain. One patient had a IDH wildtype sample with a methylation profile consistent with reactive brain tissue rather than tumour. CONCLUSIONS All four cases of IDH mutation change at glioma recurrence were due to either misinterpreting perilesional or reactive tissue as tumour or overlooking a non-canonical mutation. This finding supports the concept that IDH mutation is a fixed characteristic of gliomas and highlights the importance of accurate surgical biopsy and molecular testing at initial diagnosis.

Variability in speaker expectations of morphosyntactic mutation in Welsh

Like all modern Celtic languages Welsh exhibits initial consonant mutation with both lexical and morphosyntactic triggers. Owing to the complexity of the system and the sociolinguistic situation of Welsh, change and variation in the system seems inevitable, and evidence for change has been observed in production. Less work, however, has focused on speakers’ attitudes and expectations in perception. We used an online auditory acceptability judgement survey to investigate expectations for different morphosyntactic soft-mutation triggers. Respondents listened to sentences with canonical and non-canonical mutation patterns and used Likert scales to indicate for each sentence whether they would use the same pattern themselves and whether they would expect it from others. Almost all respondents expected some variation, even in their own production, but two main clusters of respondents could be identified: “Conservative” respondents whose expectations were close to canonical mutation patterns and “Variable” respondents whose expectations were considerably more flexible. First-language status was the only demographic variable to predict respondent attitudes, suggesting that L2 Welsh speakers accept noncanonical mutation to a greater extent than L1 Welsh speakers. We also compared different mutation triggers, with the tentative conclusion that apparently identical triggers may not in fact be identical for all speakers, and that trigger transparency may be an important factor in predicting variability.

Non-canonical genomic driver mutations of urethane carcinogenesis.

The carcinogen urethane induces pulmonary tumors in mice initiated by an incredibly specific Q61L/R oncogenic mutation in the proto-oncogene Kras. Previous Whole-Exome Sequencing of urethane-induced tumors revealed a bias towards A➙T/G and G➙A substitutions. Subsequent ultra-sensitive Maximum-Depth Sequencing of Kras shortly after urethane exposure suggest a further refinement to CA➙CT/G substitutions. As C182AA➙C182T/GA substitutions in Kras result in Q61L/R mutations, the extreme bias of urethane towards these genomic driver mutations can be ascribed to the specificity of the carcinogen for CA➙CT/G substitutions. However, we previously found that changing rare codons to common in the Kras gene to increase protein expression shifted mutations in urethane-induced tumors away from Kras, or when detected in Kras, to G12D mutations that are usually rarely detected in such tumors. Moreover, the loss of p53 partially reversed this effect, generating tumors with either Q61L/R or G12D oncogenic Kras mutations, or no Kras mutations, presumably due to other genomic driver mutations. Determining the origin of these G12D and other unknown non-canonical genomic driver mutations would provide critical insight into the extreme bias of carcinogens for specific genomic driver mutations. We thus compared the types of Single Nucleotide Variations detected by previously performed Maximum-Depth Sequencing immediately after urethane exposure to the mutation signatures derived from Whole Exome Sequencing of urethane-induced tumors. This identified two types of non-canonical mutations. First, a V637E oncogenic mutation in the proto-oncogene Braf that conforms to the mutation signature of urethane, suggesting that the mutational bias of the carcinogen may account for this non-canonical mutation, similar to that for canonical Q61L/R mutations in Kras. Second, G12D and Q61H mutations in Kras that did not fit this mutation signature, and instead shared similarity with Single Nucleotide Variations detected by Maximum-Depth Sequencing from normal cells, suggesting that perhaps these mutations were pre-existing. We thus posit that when canonical Kras mutations are selected against that the carcinogen may instead promote the expansion of pre-existing genomic driver mutations, although admittedly we cannot rule out other mechanisms. Interrogating the mutation signatures of human lung cancers similarly identified KRAS genomic driver mutations that failed to match the mutation signature of the tumor. Thus, we also speculate that the selection for non-canonical genomic driver mutations during urethane carcinogenesis may reflect the process by which discordance between genomic driver mutations and mutational signatures arises in human cancers.

PATH-15. NON-CANONICAL IDH 1 AND IDH 2 MUTATIONS ARE ASSOCIATED WITH IMPROVED SURVIVAL IN PATIENTS WITH GLIOMAS: RESULTS OF A META-ANALYSIS

Abstract BACKGROUND Non-canonical isocitrate dehydrogenase (IDH) mutations are uncommon among patients with grade 2 and 3 gliomas and their clinical significance is still unclear. METHOD We carried out a systematic review and meta-analysis to evaluate the prognostic impact of IDH1 and IDH2 non-canonical mutations. We searched English-written articles published on PubMed/Medline, Cochrane library, and Scopus until the 1st May 2021. Keywords adopted for the research were: ‘’IDH’’ OR ‘’IDH1’’ OR ‘’IDH2’’ OR ‘’Isocitrate dehydrogenase’’ AND ‘’glioma’’. RESULTS We selected 13 of 3513 studies reporting data of 4007 patients with a diagnosis of grade 2 and grade 3 including 3091 patients with a molecular proven IDH 1 or IDH 2 mutations. Between patients with IDH mutated gliomas, 479 (15.5%) patients showed non-canonical IDH 1 or IDH 2 mutations. The presence of IDH 1 non-canonical mutation occurred in younger age and in non codeleted 1p19q as compared to canonical IDH 1 mutation. However, patients with IDH 2 mutations showed more frequently 1p19q codeletion as compared to those with canonical IDH 1 mutated glioma. Four studies reported complete data survival for patients with non-canonical (n= 160) and canonical mutations (n= 1019). The pooled HR of these studies was 0.47 (95% CI, 0.28-0.81) confirming a positive prognostic role for non-canonical IDH 1 and IDH 2 mutations. CONCLUSION The presence of non-canonical IDH 1 and IDH 2 mutations defines a specific subgroup of gliomas occurring at younger age with improved survival. Patients with a non-canonical IDH 1 mutation showed less frequently 1p19q codeletion as compared to those patients harboring a canonical or IDH 2 mutation.

Synchronous supratentorial and infratentorial oligodendrogliomas with incongruous IDH1 mutations, a case report

Infratentorial oligodendrogliomas, a rare pathological entity, are generally considered metastatic lesions from supratentorial primary tumors. Here, we report the case of a 23-year-old man presenting with a histopathologically confirmed right precentral gyrus grade 2 oligodendroglioma and a concurrent pontine grade 3 oligodendroglioma. The pontine lesion was biopsied approximately a year after the biopsy of the precentral lesion due to disease progression despite 4 cycles of procarbazine-CCNU-vincristine (PCV) chemotherapy and stable supratentorial disease. Histology and genetic analysis of the pontine biopsy were consistent with grade 3 oligodendroglioma, and comparison of the two lesions demonstrated common 1p/19q co-deletions and TERT promoter mutations but distinct IDH1 mutations, with a non-canonical IDH1 R132G mutation identified in the infratentorial lesion and a R132H mutation identified in the cortical lesion. Initiation of Temozolomide led to complete response of the supratentorial lesion and durable disease control, while Temozolomide with subsequent radiation therapy of 54 Gy in 30 fractions resulted in partial response of the pontine lesion. This case report supports possible distinct molecular pathogenesis in supratentorial and infratentorial oligodendrogliomas and raises questions about the role of different IDH1 mutant isoforms in explaining treatment resistance to different chemotherapy regimens. Importantly, this case suggests that biopsies of all radiographic lesions, when feasible and safe, should be considered in order to adequately guide management in multicentric oligodendrogliomas.

Novel Therapeutic Targets for DNMT3A Mutant Myeloid Neoplasms

The DNMT3A mutations (DNMT3AMT) that occur in acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) are either hypomorphic or inactivating, consistent with bone fide tumor suppression function of DNMT3A . The most recurrent mutation involves the canonical R882, resulting in a hypomorphic protein most likely also acting in a dominant-negative manner by affecting the methyltransferase activity of the remaining wild type DNMT3A allele (DNMT3AWT).To assess the clinical context of DNMT3A alterations, 1059 samples from patients with myeloid neoplasia (422 MDS, 240 MDS/myeloproliferative neoplasms (MPN), 163 secondary (s) AML, 234 primary (p) AML were examined. DNMT3AMT were found in 21% of patients, of which 71% were missense mutations [52% R882; 48% non-canonical mutation (NCM)] and 29% were truncating mutations or frameshift mutations. DNMT3AR882 was significantly overrepresented in pAML (P=.04) and underrepresented in MDS (P=.03). In contrast, truncating mutations were more common in MDS and sAML vs. pAML (55% vs. 29%; P<.01). DNMT3AMT were associated with ASXL1 (P=.05) and SF3B1 (P<.01) mutations in MDS but not in pAML. In contrast, DNMT3AMT associated with NPM1 (P<.01), FLT3ITD/TKD (P=.04) and IDH1 mutations in pAML but not sAML. Clonal architecture analyses showed that 49% of R882 were ancestral events with TET2, FLT3ITD/TKD and NPM1 as the most common secondary hits. In contrast, only 33% of non-R882 variants (39% truncating; 33% NCM) were an ancestral event (P=.04). Gene expression analyses of AML [n=167 (26% DNMT3AMT)] and MDS [n=175 (8% DNMT3AMT)] showed a decrease in DNMT3A mRNA levels in DNMT3AMTvs. DNMT3AWT AML (mean ± SD= 1079 ± 582 vs. 1376 ± 556), though not in MDS, probably due to the high percentage of truncating mutations and resulting nonsense mediated decay of mRNA.Exclusively heterozygous DNMT3AMT configurations imply that biallelic DNMT3A loss may be lethal and thus that mutant cells may be amenable to synthetic lethal therapeutic approaches. DNMT3A inhibitors may however, also phenocopy DNMT3A loss in normal DNMT3AWT cells. Although such concerns are not huge, as they would likely be reversed by withdrawing the inhibitor, there is also rationale to instead overcome haploinsufficiency by increasing the throughput of the remaining DNMT3AWT allele, i.e. to revert malignant cells toward normalcy rather than attempt to kill them. We achieve this by supplementation of the universal methyl donor S-adenosyl- L -methionine (SAM), i.e. by increasing DNMT3A substrate availability.To test this principle we quantified the differential sensitivity of DNMT3AMTvs. DNMT3AWT and primary cells to SAM (disulfate tosylate) supplementation. As in vitro models, we selected a myeloid derived cell line, OCI-AML3, which harbors R882 and shows a 9% decrease in global DNA-methylation. As controls we used a panel of myeloid cell lines with DNMT3AWT (K562, THP1, U937, MOLM13). We observed that OCI-AML3R882 exhibited higher sensitivity to 24h SAM supplementation than DNMT3AWT cell lines using an MTT assay (IC50 of ~2mM). This dose did not affect healthy human bone marrow cells as shown by no change in growth upon SAM treatment.We then assessed DNMT3A enzymatic activity using an ELISA-like colorimetric assay and found that 2mM SAM exposures increased DNMT3A activity by 20% vs. baseline (P=.04) in OCI-AML3R882 cells, suggesting that SAM supplementation was able to increase DNMT3AWT throughput. DNMT3AWT cells did not show increases in DNMT3A activity with SAM supplementation.We also generated a cellular model of DNMT3A insufficiency using lentiviral shRNA in K562 (55% reduction by qPCR) (K562-shRNA DNMT3A). SAM treatment (500µM, 1mM, 2mM, 3mM) induced a decrease in cell growth in K562-shRNA DNMT3A vs. not (30% vs. 60%; P<.01).In sum, we present the first novel DNMT3AMT therapeutic strategy based on the principle of overcoming the functional deficiency of a monoallelic mutants through up-modulation of DNMT3AWT activity. SAM showed a promising increase in DNMT3AWT activity in mutant cells at safe/feasible doses, opening the possibility of combinational strategies with existing/ approved agents. SAM could be beneficial in healthy older adults with clonal hematopoiesis of indeterminate potential and DNMT3AMT. We are in the process of determining if SAM treatment will enhance DNMT3AWT in murine models of DNMT3A (Dnmt3aMT, Flt3ITD, Npm1C) resulting in a fully penetrant leukemic phenotype. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

IDH1 Non-Canonical Mutations and Survival in Patients with Glioma.

Background: Non-canonical mutations of the isocitrate dehydrogenase (IDH) genes have been described in about 20–25% and 5–12% of patients with WHO grade II and III gliomas, respectively. To date, the prognostic value of these rare mutations is still a topic of debate. Methods: We selected patients with WHO grade II and III gliomas and IDH1 mutations with available tissue samples for next-generation sequencing. The clinical outcomes and baseline behaviors of patients with canonical IDH1 R132H and non-canonical IDH1 mutations were compared. Results: We evaluated 433 patients harboring IDH1 mutations. Three hundred and ninety patients (90.1%) had a canonical IDH1 R132H mutation while 43 patients (9.9%) had a non-canonical IDH1 mutation. Compared to those with the IDH1 canonical mutation, patients with non-canonical mutations were younger (p < 0.001) and less frequently presented the 1p19q codeletion (p = 0.017). Multivariate analysis confirmed that the extension of surgery (p = 0.003), the presence of the 1p19q codeletion (p = 0.001), and the presence of a non-canonical mutation (p = 0.041) were variables correlated with improved overall survival. Conclusion: the presence of non-canonical IDH1 mutations could be associated with improved survival among patients with IDH1 mutated grade II–III glioma.

Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma

SUMMARYSomatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic “driver” mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions. These mutations display transcriptional asymmetry that is modulated by nucleotide excision repair (NER), indicating that they are caused by UV photoproducts. Using a sequencing method called UV DNA endonuclease sequencing (UVDE-seq), we confirm the existence of an atypical thymine-adenine photoproduct likely responsible for UV-induced T>A substitutions. Similar non-canonical mutations are present in skin cancers, which also display transcriptional asymmetry and dependence on NER. These include multiple driver mutations, most prominently the recurrent BRAF V600E and V600K substitutions, suggesting that mutations arising from rare, atypical UV photoproducts may play a role in melanomagenesis.

4373 Defining the role of non-canonical PIK3CA mutations in head and neck squamous cell carcinoma

OBJECTIVES/GOALS: To characterize the oncogenic potential of HNSCC cell lines harboring 17 non-canonical PIK3CA mutations. METHODS/STUDY POPULATION: Non-canonical PIK3CA mutant constructs generated via site-directed mutagenesis are subcloned into doxycycline-inducible vector pLVX-Puro. Serum-dependent HNSCC cell line (PCI-52-SD1) is then stably transfected with vectors and undergo doxycycline-induction. Cell survival is determined by depriving cells of fetal bovine serum for 72 hours and quantifying remaining cells with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell proliferation and migration is evaluated with colony formation assays and transwell assays respectively. RESULTS/ANTICIPATED RESULTS: To date, the survival behavior of eight non-canonical mutants was assessed. Three mutants – Q75E, V71I, and E970K – exhibited 18.7-26.7% greater survival rate relative to cells transfected with wild-type. Five mutants – R519G, Y606C, W328S, C905S, and M1040I – demonstrated survival rates that differed only by −4.3% to +6.6% relative to wild-type. We hypothesize the three activating mutants that exhibited increased survival will also demonstrate increased cell proliferation and migratory behavior whereas the three neutral mutants will not differ from control. DISCUSSION/SIGNIFICANCE OF IMPACT: Ongoing HNSCC PI3K inhibitor trials could be more effective if all PIK3CA hyperactivation mutations are known. Identifying non-canonical mutation effects could result in greater efficacy if drugs are restricted only to those with activating mutations. CONFLICT OF INTEREST DESCRIPTION: JRG and DEJ are co-inventors of cyclic STAT3 decoy and have financial interests in STAT3 Therapeutics, Inc. STAT3 Therapeutics, Inc. holds an interest in a cyclic STAT3 decoy oligonucleotide. The remaining authors declare no conflicts.

Analysis of canonical and noncanonical splicing site mutation of MET that causes exon 14 skipping.

e21513 Background: The hepatocyte growth factor receptor gene ( MET) exon 14 skipping ( METex14) has been wildly accepted as a driver alteration in lung cancer targetable by tyrosine kinase inhibitors (TKIs) such as crizotinib. While it is easy to interpret canonical splicing site mutations, it is more controversial to interpret noncanonical splicing site mutations. Methods: Hybrid capture–based next generation sequencing of 59-1021 genes including MET was performed at the request of individual treating physicians. The mutation profiling of MET were retrospectively analyzed. Results: Of 3500 lung cancers profiled by comprehensive genomic profiling, 211 harbored MET single nucleotide mutation (SNV), or small insertion/deletion (InDel). Among which 66 cases were predicted to have METex14. Forty-two (62.1%) cases harbored mutations affecting the splicing donor site, while 24 harbored mutations affecting the splicing acceptor site. Thirty-four of the 42 cases affecting the splicing donor site had mutations in the canonical splicing site, including 3 cases harbored MET c.3028G &gt; A/T/C mutation, 14 cases harbored c.3028+1G &gt; A/T/C, 10 cases harbored c.3028+2T &gt; A/C/G, 8 cases harbored deletion of the whole canonical splicing site. Interestingly, all the seven cases affecting the noncanonical splicing donor site were c.3028+3A &gt; G/T mutation. On the contrary, all the 24 mutations affecting the splicing acceptor site were deletion with/without insertion, with 12 affecting the canonical site and 12 the noncanonical site. We speculated that the polypyrimidine tract around the splicing acceptor site making it more susceptible to deletion/insertion, and thus single nucleotide variant less common. Conclusions: At the 5’ end of the exon 14 of MET, most of the genome alterations are deletions with/without insertions affecting the canonical and noncanonical splicing acceptor site. At the 3’ end, they are mostly point mutations affecting the splice donor site. The noncanonical splicing donor site c.3028+3A plays crucial role in the splicing of exon 14 in MET.

Elevation of Urinary 2-Hydroxyglutarate in IDH-Mutant Glioma.

Recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, which are frequent in gliomas, result in marked accumulation of the metabolic by-product 2-hydroxyglutarate (2-HG) within tumors. In other malignancies, such as acute myeloid leukemia, presence of IDH mutation is associated with elevated 2-HG levels in serum or urine compartments. Circulating 2-HG in patients with glial malignancies has not been thoroughly investigated. In this study, we analyzed 2-HG levels in the serum and urine of a large set of patients with IDH-mutant and IDH-wild-type glioma, and the cerebrospinal fluid (CSF) from a subset of this cohort. We found that 2-HG was elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma, although no significant differences in 2-HG levels were observed in the serum or the small set of CSF samples obtained. Among patients with IDH-mutant glioma, 2-HG levels did not differ based on the histopathologic grade, genetic subtype (TP53 mutant or 1p/19q codeleted), presence of a canonical (IDH1 R132H) or noncanonical (any other IDH variant) mutation, or treatment type. Our finding suggests that urinary 2-HG is increased among patients with IDH-mutant gliomas, and may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management. Patients with glioma who harbor mutations in isocitrate dehydrogenase genes showed selective elevation of the oncometabolite 2-hydroxyglutarate in the urine. Similar elevations were not identified in the serum or cerebrospinal fluid. 2-Hydroxyglutarate may serve as a useful, noninvasive biomarker to stratify patients newly diagnosed with glioma with regard to prognosis and management.