Hotspot Point Mutations Research Articles

Pseudoendocrine sarcoma: a rare new entity with unique radiologic and pathologic/molecular characteristics.

Pseudoendocrine sarcoma is a rare, recently described intermediate grade sarcoma of uncertain phenotypethat most commonly affects theparaspinal location in older patients with a distinctive endocrine/paraganglioma-like morphology and unique CTNNB1 point mutation. While these tumors appear as epithelial or even benign endocrine tumors, these lack markers for such and are highlighted by nuclear expression of beta-catenin. This case is the first among the previously reported only twenty-five cases of this entity,including one original series and a few case reports, to correlate the radiologic imaging with the pathologic features. Furthermore, this case illustrates the oldest-to-date patient with this unique location as a palpable painful chest wall/paraspinal location, with new morphologic observations and, finally, this isonly the second case to havethisspecific CTNNB1 hotspot point mutation for this rare entity.

A comprehensive genomic study of 390 H3F3A-mutant pediatric and adult diffuse high-grade gliomas, CNS WHO grade 4.

Malignant brain tumors, known as H3K27-altered diffuse midline glioma (DMG) and H3G34-mutant diffuse hemispheric glioma (DHG), can affect individuals of all ages and are classified as CNS WHO grade 4. We comprehensively characterized 390 H3F3A-mutant diffuse gliomas (201 females, 189 males) arising in pediatric patients (under 20years old) and adults (20years and older) evaluated by the CGP program at Foundation Medicine between 2013 and 2020. We assessed information from pathology reports, histopathology review, and clinical data. The cohort included 304 H3K27M-mutant DMG (156 females, 148 males) and 86 H3G34-mutant DHG (45 females, 41 males). Median patient age was 20years (1-74years). The frequency of H3K27M-mutant DMG was similar in both pediatric and adult patients in our cohort-48.6% of the patients were over 20years old, 31.5% over 30, and 18% over 40 at initial diagnosis. FGFR1 hotspot point mutations (N546K and K656E) were exclusively identified in H3K27M-mutant DMG tumors (64/304, 21%; p = 0.0001); these tend tooccur in older patients (median age: 32.5years) and mainly arose in the diencephalon. H3K27M-mutant DMG had higher rates of mutations in NF1 (31.0 vs 8.1%; p = 0.0001) and PIK3CA/PIK3R1 (27.9% vs 15.1%; p = 0.016) compared to H3G34-mutant DHG. However, H3G34-mutant DHG had higher rates of targetable alterations in cell-cycle pathway genes (CDK4 and CDK6 amplification; CDKN2A/B deletion) (27.0 vs 9.0%). Potentially targetable PDGFRA alterations were identified in ~ 20% of both H3G34-mutant DHG and H3K27M-mutant DMG. Overall, in the present study H3K27M-mutant DMG occurred at similar rates in both adult and patient patients. Through our analysis, we were able to identify molecular features characteristic of DMG and DHG. By identifying the recurrent co-mutations including actionable FGFR1 point mutations found in nearly one-third of H3K27M-mutant DMG in young adults, our findings can inform clinical translational studies, patient diagnosis, and clinical trial design.

Abstract LB555: Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress

Abstract Clonal hematopoiesis (CH), the age-related expansion of specific clones in the blood system has been considered as a pre-malignant condition of blood cancers. Approximately 50% of CH manifests from DNMT3A mutations acquired in the hematopoietic stem cells (HSCs), suggesting these mutations may convey a fitness advantage to HSCs during hematopoietic stress. However, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations enhances HSC fitness under recurrent inflammatory stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated by specifically deleting Dnmt3a heterozygously (Vav-Cre;Dnmt3afl/+ = Dnmt3aHET) or homozygously (Vav-Cre;Dnmt3afl/fl = Dnmt3aKO) from the hematopoietic system. A hematopoietic system-specific knockin model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre;Dnmt3aR878H/+ = Dnmt3aR878) was also included in the analysis. Competitive transplantation assays coupled with inflammatory and proliferative challenges suggested that Dnmt3aKO and Dnmt3aR878 HSCs were specifically tolerant of the deleterious effects of IFNg on HSC self-renewal and clonal expansion. When the competition between Dnmt3a-mutant and control HSCs under IFNg exposure was directly quantified in a novel mouse model, we found Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Mechanistically, DNA hypomethylation-associated over-expression of Txnip in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs lead to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence due to a prolonged G0 exit in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not control 32D cells. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation ex vivo. In vivo, down-regulation of p21 had no effect on control HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-associated exhaustion in a transplantation experiment. In summary, our data suggest an augmented Txnip-p53-p21 axis preserves the functional potential of Dnmt3a-mutant HSCs under inflammatory stress, which highlights a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. Citation Format: Christine R. Zhang, Elizabeth Ostrander, Won Kyun Koh, Ostap kukhar, Hamza Celik, Jeffrey Magee, Grant Challen. Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress [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 LB555.

Characterization of molecular and immune landscape of microvascular invasion in hepatocellular carcinoma and prognostic significance.

e16162 Background: Partial hepatectomy is potentially curative treatment in selected patients (pts) with hepatocellular carcinoma (HCC). Microvascular invasion (MVI) is one of the strongest predictors of prognosis and recurrence of HCC. In this study, we analyzed the gene mutation signature and immune factors associated with MVI, and the prognostic value. Methods: Pts who were diagnosed with HCC between Jan 2018 and Sep 2020 in Cancer Hospital of Chinese Academy of Medical Sciences were retrospectively enrolled in this study. Next-generation sequencing (NGS) was performed on tumor tissues after radical partial hepatectomy. The different immune makers (PD1, PD-L1, CD3, CD8, CD68, and CD163) of tumor-infiltrating lymphocytes (TILs) were identified with immunofluorescence-based microenvironment analysis panel (MAP). Results: In the MVI+ group (n = 39, 59.1 %), there were significantly more pts with larger tumor size, and capsules invasion by tumors. The mutation spectra revealed that 61 (92.4%) pts harbored somatic mutations and the most frequently mutated gene was telomerase reverse transcriptase (TERT), which was present in 31.8% (n = 20) HCC pts. Mutations in the promoter of TERT were found in 95.2% (20/21) pts. 90.0% (18/20) had one hotspot point mutation located 124bp upstream of the translation start site. The mutation frequency in the TERT promoter was significantly higher in the MVI+ group compared with the MVI- group (41.0% vs. 14.8%, p < 0.05). Pts with TERT promoter mutations tend to be MVI+ rather than MVI- and more HCC driver mutations were detected in MVI+ pts with TERT promoter mutations (p < 0.05). Based on MAP, we found MVI+ pts had significantly lower CD3+ TILs (p < 0.05). The multivariable analysis indicated that pts with copy number variations (CNV, hazard ratios [HR] 2.62, 95% confidence interval [CI] 1.07-6.40; p = 0.028), elevated PT/INR (HR 0.29, 95% CI 0.11-0.77; p = 0.008), and lower serum prealbumin concentration (PALB, HR 3.03, 95% CI 1.08-8.51; p = 0.027) had significantly shorter recurrence-free survival (RFS) after surgery. Similar findings were also observed for immune factors within cancer parenchyma, including CD68+ (HR 7.14, 95% CI 0.95-53.55; p = 0.025), CD163+ (HR 3.07, 95% CI 1.10-8.53; p = 0.023), CD68+/CD163- (HR 11.35, 95% CI 1.51-85.05; p = 0.003), CD68+/CD163+ (HR 3.51, 95% CI 1.4-8.79; p = 0.004), which indicated tumor-associated macrophage (TAM) infiltration was independent risk factor for RFS. Conclusions: In this study, we characterized the molecular and immune landscape of MVI in HCC patients. MVI presence highly correlated with adverse biological markers, mutations in the promoter of TERT, and higher CD3+ TILs. Some risk factors of HCC recurrence were identified, including CNV, PT/INR, CA 19-9, PALB, and TAM. Transcriptional alterations and immune tumor microenvironment change may contribute to MVI in HCC.

Txnip enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells Via p21

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Approximately 50% of CH variants occur in the gene DNMT3A. While DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under recurrent hematopoietic stress.To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated; Dnmt3a heterozygous (Vav-Cre; Dnmt3afl/+ = Dnmt3aHET) and homozygous (Vav-Cre; Dnmt3afl/fl = Dnmt3aKO) hematopoietic loss-of-function, and a knock-in model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre; Dnmt3aR878H/+ = Dnmt3aR878).When Dnmt3a-mutant cells were competitively transplanted with wild-type (WT) competitor bone marrow (BM) cells and challenged with different inflammatory and proliferative stressors, Dnmt3aKO and Dnmt3aR878 HSCs were specifically resistant to the deleterious effects of IFNg on HSC self-renewal and clonal expansion. This insensitivity was also confirmed in a humanized mouse model where human CD34 + cord blood cells edited with DNMT3A-targeting gRNAs were xenografted into recipient mice and episodically exposed to human recombinant IFNg. DNMT3A mutant cells maintained their clone size, whereas AAVS1-targeted cells (control) were depleted over serial transplantation. These data suggest that Dnmt3a-mutant HSCs, mimicked DNMT3A-mutated human HSCs and are specifically resistant to IFNg-mediated depletion. One explanation for the observed resistance is that Dnmt3a-mutant HSCs have a fitness advantage under IFNg challenge. Therefore, we generated a novel mouse model to directly quantify the competition between Dnmt3a-mutant and WT HSCs. 10% donor BM cells (CD45.2; WT or Dnmt3a-mutant), 10% WT competitor BM cells (CD45.1/2; Ubc-GFP+) and 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA-IFNg) that express IFNg by doxycycline were transplanted into CD45.1 recipient mice. To normalize the effect of doxycycline, chimera made with 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA) were also transplanted into recipients. Our result from this transplantation experiment showed Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Genetic ablation of IFNgr1 from Dnmt3a-mutant mice revealed that IFNg signaling is cell-intrinsically required by clonal expansion of Dnmt3a-mutant HSCs. In parallel, when HSCs were transplanted into IFNg-deficient recipient mice, clonal expansion of Dnmt3aKO HSCs but not WT HSCs was significantly compromised, suggesting IFNg signaling is also cell-extrinsically crucial for the clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated over-expression of Thioredoxin-interacting protein (Txnip) in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs led to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence and resistance to apoptosis in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not WT 32D murine myeloid cell line. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 re-sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation and apoptosis. In vivo, down-regulation of p21 had no effect on WT HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-induced exhaustion in a transplantation experiment.Taken together, our data highlighted a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which suggests a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Present and Future of Less-invasive Liquid Biopsy for the Diagnosis of Gliomas and Brain Tumors

There is growing interest in liquid biopsy, the less-invasive detection of circulating tumor DNA(ctDNA)or circulating tumor cells(CTCs)from cerebrospinal fluid(CSF)and/or serum of patients, for the diagnosis of brain tumors. We share our experience of detecting hot spot point mutations using droplet digital PCR(ddPCR)in ctDNA obtained from the CSF of patients with brain tumors. The detection of mutations such as IDH1 R132H, BRAF V600E, and TERT promoter mutations in gliomas can be diagnostic. For optimal detection of ctDNA, which is only seen at very low concentrations, proper handling and storage of CSF, high-yield extraction of ctDNA, and usage of sensitive PCR methods for detection are imperative. We discuss which mutations can be assessed when diagnosing brain tumors, with a specific focus on gliomas. Finally, we look at what the near future holds for liquid biopsy of brain tumor patients, including next-generation sequencing panel analysis and accurate assessment of fusion genes.

Significance of BRAFV600E mutation in intra-axial brain tumor in Malaysian patients: case series and literature review.

To date, BRAF mutations in brain tumor patients have not been characterized in the Malaysian population. Based on the numerous reported studies, there are main mutations that exist in BRAF gene in various types of cancers. A missense mutation in codon 600of the BRAF nuclear oncogene (BRAFV600E) is the most prevalent hotspot point mutation that has been identified in multiple human malignancies. We here aimed to find out the frequency of BRAFV600E mutation in a series of Malaysian patients with brain tumors and if any association exists between BRAFV600E mutation and clinicopathological features of patients. Fresh frozen tumor tissue samples from 50Malaysian brain tumor patients were analyzed for BRAFV600E mutational status, and its correlation with clinicopathological features (including age, gender, and tumor localization such as intra-axial: within the brain substance or extra-axial: outside the brain substance) was examined. The overall BRAFV600E mutation frequency was determined to be 22% (in 11of 50patients). BRAFV600E was significantly correlated with the tumor location group, which shows BRAFV600E was more frequent in the intra-axial tumor than the extra-axial tumor group. In this study, we also observed that male patients were slightly more susceptible to BRAFV600E mutation, and this mutation was predominant in patients of the age group<40years. However, these parameters did not translate to statistical significance. The data demonstrate that BRAFV600E mutation is observed significantly more often in intra-axial brain tumor patients, which can serve as baseline information for further research on genetic alteration that occurs during brain tumor progression in the Malaysian population.

XBP1 increases transactivation of somatic mutants of ESR1 and loss of XBP1 reverses endocrine resistance conferred by gain-of-function Y537S ESR1 mutation

Somatic mutations of the estrogen receptor 1 gene (ESR1) is an emerging mechanism of acquired resistance to endocrine therapy in hormonal breast cancers. Hotspot point mutations in the ligand- binding domain of estrogen receptor α (ER) and genomic rearrangements producing ESR1 fusion genes are the two major types of mutations that are associated with endocrine resistance. The crosstalk between X-box binding protein 1 (XBP1), a key transcription factor of the unfolded protein response (UPR) and ER signalling creates a positive feedback loop that results in increased expression of XBP1 in ER-positive breast cancer. Here we report that XBP1 co-operated with point mutants (Y537S, D538G) and fusion mutants (ESR1-YAP1, ESR1-DAB2) of ESR1 to increase their transcriptional activity. XBP1 was required for optimal expression of estrogen-regulated genes, and up to 40% of XBP1-dependent genes were estrogen-responsive genes. Knockdown of XBP1 in genome-edited MCF7 cells expressing Y537S mutant reduced their growth, re-sensitized them to anti-estrogens and attenuated the constitutive and estrogen-stimulated expression of estrogen-regulated genes. Our study provides a rationale for overcoming endocrine resistance in breast cancers expressing ESR1 mutation by combining the XBP1 targeting agents with anti-estrogen agents.

Abstract 1810: The mutant IDH1 inhibitor ivosidenib promotes tumor cell differentiation and anti-tumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (ICC) is a cancer of the liver bile ducts, which has been rising in incidence and carries a 5-year survival rate of less than 10%. Mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2) are among the most common genetic alterations in ICC, present in 20% patients. The hot-spot point mutations of IDH result in aberrant enzymatic function, reducing alpha-ketoglutarate (alphaKG) to the oncometabolite, 2-hydroxyglutarate (2-HG), which accumulates to millimolar concentrations within the cell. 2-HG competitively inhibits alphaKG-dependent histone and DNA demethylating enzymes, thereby altering chromatin regulation. The first-in-class small molecule selective IDH1m inhibitor, ivosidenib, has shown benefit in phase I and III trials for patients with IDH1m ICC. In order to gain insight into the mechanisms underlying the response to IDH1m inhibition, we developed the first genetically engineered mouse model (GEMM) of IDH1m ICC. We found that ivosidenib reduced the overall tumor burden and increased overall survival of syngeneic allografts of ICCs derived from the GEMM. The positive response to ivosidenib was associated with an induction of mature hepatocyte lineage markers and a reduction of tumor produced 2-HG &amp;gt;85%, supporting observations from the ivosidenib phase I data in ICC patients that mIDH1 inhibition may promote ICC tumor cell differentiation. In addition, there was pronounced induction of immune stimulatory interferon signaling, increased recruitment of CD8+ T cells to the tumors and strong PD-L1 upregulation. Thus, ivosidenib treatment leads to a sustained inhibition of 2-HG production, which correlated with an increased immune stimulation compared to vehicle treatment. We will present these data as well as ongoing studies interrogating the immune and transcriptomic changes with further resolution via fluorescence-activated cell sorting and single-cell RNA-sequencing analysis, direct functional testing of contributions of specific immune populations to the therapeutic efficacy of ivosidenib, and testing ivosidenib in combination with checkpoint inhibition. These studies highlight the impact of ivosidenib on hepatocyte differentiation and immune stimulation in IDH1m ICC and support the key role of IDHm in ICC tumor maintenance. Citation Format: Meng-Ju Wu, Lei Shi, Juan Dubrot, Christine Hudson, Joshua Merritt, Ramzi Adil, Qibiao Wu, Sebastien Ronseaux, Rohini Narayanaswamy, Robert Manguso, Brandon Nicolay, Nabeel Bardeesy. The mutant IDH1 inhibitor ivosidenib promotes tumor cell differentiation and anti-tumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1810.

An enzymatic on/off switch-mediated assay for KRAS hotspot point mutation detection of circulating tumor DNA.

BackgroundTo detect the mutations of KRAS gene in colorectal cancer patients and other cancer patients, it is of value to develop non‐invasive, sensitive, specific, easy, and low‐cost assays.MethodsTemplates harboring hotspot mutations of the KRAS gene were constructed, and primers were designed for evaluation of the specificity, and sensitivity of detection system consisted of exonuclease polymerase‐mediated on/off switch; then, gel electrophoresis and real‐time PCR were performed for verification. The assay was verified by testing the DNA pool of normal controls and circulating DNA (ctDNA) samples from 14 tumor patients, as compared to Sanger sequencing.ResultsA specific and sensitive assay consisted of exonuclease polymerase‐mediated on/off switch, and multiplex real‐time PCR method has been established. This assay could detect <100 copies of KRAS mutation in more than 10 million copies of wild‐type KRAS gene fragments. This assay was applied to test KRAS gene mutations in three cases of fourteen ctDNA samples, and the results were consistent with Sanger sequencing. However, this PCR‐based assay was more sensitive and easier to be interpreted.ConclusionThis assay can detect the presence of KRAS hotspot mutations in clinical circulating tumor DNA samples. The assay has a potential to be used in early diagnosis of colorectal cancer as well as other types of cancer.

AKR1D1 and CYP7B1 mutations in patients with inborn errors of bile acid metabolism: Possibly underdiagnosed diseases

Inborn errors of bile acid metabolism (IEBAM) cause rare but treatable genetic disorders that can present as neonatal cholestasis or neurological diseases. Without timely primary bile acid treatment, patients may develop liver failure early in life. This study aimed to analyze the types and treatment outcomes of IEBAM in Taiwanese infants and document the allele frequency of CYP7B1 hot spot mutations in the population. Urine samples from patients with infantile intrahepatic cholestasis and suspected IEBAM were subjected to urinary bile acid analysis by gas chromatography-mass spectrometry (GC/MS). Genetic diagnoses were made using direct sequencing or next-generation sequencing. We also tested healthy control subjects for a probable hot spot point mutation of CYP7B1. Among the 75 patients with infantile intrahepatic cholestasis tested during 2000 -2016, three had ∆4-3-oxosteroid 5β-reductase deficiency with AKR1D1 mutations, and three had oxysterol-7α-hydroxylase deficiency with CYP7B1 mutation. Two patients with ∆4-3-oxosteroid 5β-reductase deficiency were successfully treated with cholic acid. The three unrelated infants with oxysterol 7α-hydroxylase deficiencies had the same p.R112X homozygous CYP7B1 mutation. Two had mild renal or neurological involvement. Among 608 healthy control subjects, the allele frequency of the heterozygous mutation for p.R112X was 2/1216 (0.16%). The only surviving patient with oxysterol 7α-hydroxylase deficiency recovered from liver failure after chenodeoxycholic acid (CDCA) treatment beginning at 3 months of age. Distinct types of IEBAM disease were found in the Taiwanese population. Patients with early diagnosis and early treatment had a favorable outcome. IEBAM prevalence rates may be higher than expected due to the presence of heterozygous mutations in the general population.

Impact of Multiple Single-Nucleotide Polymorphisms Within mprF on Daptomycin Resistance in Staphylococcus aureus.

A number of single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been associated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. Such SNPs have been found throughout the mprF ORF, although there are clearly preferred "hot spots" within this gene frequently linked to DAP-R phenotype. These mprF SNPs are often correlated with a gain-in-function phenotype, either in terms of increased production (synthase activity) and/or enhanced translocation (translocase activity) of lysyl-phosphatidylglycerol (L-PG) within its cell membrane. However, it is unclear if multiple hot spot mprF SNPs can accumulate within mprF ORFs and cause additive elevations of DAP minimum inhibitory concentrations (MICs). In this study, we used a previously well-characterized plasmid complementation system in S. aureus Newman ΔmprF mutant to express: (1) single point-mutated forms of mprF ORFs cloned from two DAP-R S. aureus strains (mprFS295L or mprFT345A) and (2) dual point-mutated forms of mprF ORFs simultaneously harboring SNPs in the central bifunctional domain and synthase domain in MprF, respectively (mprFS295L+L826F or mprFT345A+L826F). The current study revealed that, although individual hot spot point mutations within mprF ORF can recapitulate signature DAP-R-associated phenotypes (i.e., increased DAP MICs, enhanced surface positive charge, and increased L-PG synthesis), accumulation of such hot spot point mutations paradoxically caused reduction in these latter three metrics.

A Novel Assay Coupling Dephosphorylation and Blue/White Colony Screening for the G > A Hotspot Mutation at Codon 13 of KRAS Gene.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.

MassARRAY-based simultaneous detection of hotspot somatic mutations and recurrent fusion genes in papillary thyroid carcinoma: the PTC-MA assay

PurposeWe exploited the MassARRAY (MA) genotyping platform to develop the “PTC-MA assay”, which allows the simultaneous detection of 13 hotspot mutations, in the BRAF, KRAS, NRAS, HRAS, TERT, AKT1, PIK3CA, and EIF1AX genes, and six recurrent genetic rearrangements, involving the RET and TRK genes in papillary thyroid cancer (PTC).MethodsThe assay was developed using DNA and cDNA from 12 frozen and 11 formalin-fixed paraffin embedded samples from 23 PTC cases, together with positive and negative controls.ResultsThe PTC-MA assay displays high sensitivity towards point mutations and gene rearrangements, detecting their presence at frequencies as low as 5%. Moreover, this technique allows quantification of the mutated alleles identified at each tested locus.ConclusionsThe PTC-MA assay is a novel MA test, which is able to detect fusion genes generated by genomic rearrangements concomitantly with the analysis of hotspot point mutations, thus allowing the evaluation of key diagnostic, prognostic, and therapeutic markers of PTC in a single experiment without any informatics analysis. As the assay is sensitive, robust, easily achievable, and affordable, it is suitable for the diagnostic practice. Finally, the PTC-MA assay can be easily implemented and updated by adding novel genetic markers, according to clinical requirements.

Abstract 139: M2698, a novel dual inhibitor of p70S6K and Akt: preclinical efficacy in gastric cancer

Abstract M2698 is a selective, ATP-competitive dual inhibitor of p70S6K and Akt1/3 that is being evaluated in a phase I clinical trial in cancer patients. The dual nature of M2698 may result in improved clinical efficacy by blocking the increased AKT activity in a compensatory feedback loop induced by PI3K/AKT/mTOR (PAM) pathway inhibition. In previous preclinical studies, M2698 was shown to have potent anti-proliferative activity in vitro, and inhibit tumor growth in some xenograft models in vivo. The current study examined the effects of M2698 on cell proliferation in a panel of 13 gastric cancer cell lines, as gastric cancers often harbor mutations in PAM pathway genes that deregulate this signalling pathway. Cells were treated with M2698 at a range of concentrations and proliferation was evaluated at least twice using the WST-8 cell proliferation assay kit (Dojindo Molecular Technologies, Inc. Japan). Two cell lines, HGC-27 and IM95m, were particularly sensitive to M2698 (50% growth inhibition concentration [GI50] 84 and 160 nM, respectively). The GI50 values for all other cell lines were &amp;gt;10-fold higher. Although HGC-27 and IM95m each carry a PIK3CA hotspot point mutation, the presence of an activating PI3K pathway mutation was not the sole determinant of sensitivity, as a third PIK3CA mutant cell line, MKN1, was far less sensitive to M2698 (GI50 9.0 uM). Western blot analysis of pharmacodynamic biomarkers showed that M2698 (1 uM) blocked the PAM pathway in both sensitive and resistant cell lines, inhibiting phospho[p]-S6 and p-PRAS40, despite increased p-Akt. Some biomarkers from the PAM pathway and other signaling pathways appeared to be associated with sensitivity to M2698, but these candidate biomarkers need to be validated in a larger panel of cell lines. Treatment of HGC-27-tumor-bearing mice with M2698 (10, 20, 30 mg/kg/day) for 14 days resulted in significant tumor growth inhibition (80.2-98.6%) at Day 29 compared to treatment with vehicle (p&amp;lt;0.01). In conclusion, we have shown that gastric cancer cell lines have a range of sensitivities to M2698 and the sensitivity cannot be explained solely by genetic mutations in the PAM pathway. M2698 provides potent PAM pathway inhibition in both sensitive and resistant gastric cancer cell lines by blocking both p70S6K and Akt. M2698 significantly inhibits tumor growth in the HGC-27 xenograft model in vivo. Citation Format: Shota Fukuoka, Takashi Kojima, Yoshikatsu Koga, Mayumi Yamauchi, Masahiro Yasunaga, Yasuhiro Matsumura, Toshihiko Doi, Takayuki Yoshino, Toshio Kuronita, Anderson Clark, Brian Elenbaas, Atsuhi Ohtsu. M2698, a novel dual inhibitor of p70S6K and Akt: preclinical efficacy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2017-139

DNA damage repair and response (DDR) gene alterations (alt) and response to PD1/PDL1 blockade in platinum-treated metastatic urothelial carcinoma (mUC).

4509 Background: Somatic DDR alts are associated with increased mutation load (ML) and improved clinical outcomes for platinum-treated mUC (Teo et al, CCR 2017). We examined the relationship between DDR alts and response to PD1/PDL1 blockade. Methods: mUC pts enrolled to phase 2 trials of atezolizumab or nivolumab who had targeted exon sequencing of 410 genes (MSK-IMPACT) were identified. Pts were dichotomized based on presence of alts in a panel of 34 DDR genes. Analyses were performed based on: (1) any DDR alts and (2) deleterious DDR alts (frameshift, splice site, nonsense or Hotspot point mutations). Study endpoint was overall response (OR) per RECIST. ML was defined as total number of nonsynonymous mutations by MSK-IMPACT. Fisher exact, Wilcoxon rank sum, and stratified logistic regression were used. Results: Fifty two pts were identified (atezo: n=18, nivo: n=34). Median age was 67 years (range: 32 – 84) and majority (44) was male. Median platinum-free interval was 10.2 months (range: 0.3 – 150.4). DDR and deleterious DDR were seen in 25 (48.1%) and 14 (26.9%) pts (including 2 MSI and 1 POLE). OR rate was 46.2%. Responses were associated with DDR alts but not with age, gender, treatment, platinum-free interval or ML (table). In univariate logistic regression model, DDR status was associated with OR (p&lt;.001) while a trend was observed with ML as a continuous variable (p=.051). While DDR alts were associated with higher ML (all: p=.001, deleterious: p=.004), the effect of DDR alts on OR remained significant regardless of ML (&gt;median: p=.027; ≤median: p=.023), indicating that the effect of DDR was independent of ML. Conclusions: DDR alts appeared to be associated with OR to PD1/PDL1 blockade and should be integrated into future validation efforts along with other potential predictors of response. [Table: see text]

NSAIDs Use after Breast Cancer Diagnosis Improves Relapse-Free and Overall Survival: Retrospective Pilot Study

Objectives: The phosphatidylinositol 3-kinase (PI3K) pathway mediates key cellular functions, including growth and proliferation, and hotspot point mutations in the catalytic subunit of PI3K ( PIK3CA) are prevalent in breast cancer (30+%). Inhibition of cyclooxygenase-2 by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) downregulates PI3K signaling activity. Our study compared relapse-free survival and breast cancer-specific mortality, according to PIK3CA mutation …

Cancer-Specific Telomerase Reverse Transcriptase (TERT) Promoter Mutations: Biological and Clinical Implications.

The accumulated evidence has pointed to a key role of telomerase in carcinogenesis. As a RNA-dependent DNA polymerase, telomerase synthesizes telomeric DNA at the end of linear chromosomes, and attenuates or prevents telomere erosion associated with cell divisions. By lengthening telomeres, telomerase extends cellular life-span or even induces immortalization. Consistent with its functional activity, telomerase is silent in most human normal somatic cells while active only in germ-line, stem and other highly proliferative cells. In contrast, telomerase activation widely occurs in human cancer and the enzymatic activity is detectable in up to 90% of malignancies. Recently, hotspot point mutations in the regulatory region of the telomerase reverse transcriptase (TERT) gene, encoding the core catalytic component of telomerase, was identified as a novel mechanism to activate telomerase in cancer. This review discusses the cancer-specific TERT promoter mutations and potential biological and clinical significances.

Abstract 4374: Activation mechanism of oncogenic C-helix shifting mutations in BRAF, EGFR, and HER2

Abstract Kinase domain mutations are frequent drivers of many different types of cancer. While the effect of hotspot point mutations (such as BRAF V600E or EGFR L858R) is well described, the mechanism of deletion mutations, such as the recurrent EGFR exon 19 deletions is not fully understood. In this work, we have discovered and characterized analogous deletions in BRAF mutant patient samples of varying tumor types, with the highest frequency (15 patients) in pancreatic cancer representing ∼5% of KRAS wildtype patients in this data set. In addition, we have identified additional patients (3) with similar deletions in HER2 that have been previously reported at low frequency in breast cancer. The crystal structure of the most frequent BRAF deletion, molecular modeling of lower frequency BRAF deletions, and extensive molecular modeling in conjunction with molecular dynamic simulations of the most frequent EGFR deletion highlight the commonality of the mechanism of activation of this class of oncogenic alterations. These deletions truncate the loop between the β3 strand and αC-helix (β3-αC loop) of the kinase domain, forcing αC into the active (“in”) conformation, and constitutively activating these kinases. Similar to BRAF V600E mutations, constitutive activity of these BRAF β3-αC deletions is CRAF-independent and dimer-independent. Because these deletions genetically restrict the flexibility of this region of the kinase domain, they render these kinases resistant to inhibitors such as vemurafenib (for BRAF) or lapatinib (for EGFR and HER2) that bind in the αC “out” conformation. Functional characterization of the full spectrum of deletion lengths explains the high prevalence of 5 amino acid deletions in BRAF, EGFR, and HER2 in various cancers, with deletions of this length resulting in the strongest activation of kinase activity likely due to the optimal positioning of αC. This work exploits the selective power of oncogenic mutations to highlight a conserved mechanism of kinase activation and underscores the importance of conformation-specific kinase inhibitors to target mutationally activated kinases in cancer. Citation Format: Scott Foster, Dan Whalen, Aysegul Ozen, Matt Wongchenko, JianPing Yin, Ivana Yen, Gabriele Schaefer, John Mayfield, Juliann Chmielecki, Phil Stephens, Lee Albacker, Yibing Yan, Kyung Song, Georgia Hatzivassiliou, Charles Eigenbrot, Christine Yu, Andrey Shaw, Gerard Manning, Nicholas Skelton, Sarah Hymowitz, Shiva Malek. Activation mechanism of oncogenic C-helix shifting mutations in BRAF, EGFR, and HER2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4374.

Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas

Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10−3 to 4.6 × 10−2) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells.