High Prevalence Of Mutations Research Articles

Abstract P3-06-14: Whole exome analysis of extreme long-term survivors with metastatic breast cancer

Abstract Background: Metastatic breast cancer (MBC) is incurable, yet some extreme long-term survivors (ELTS) live for a decade or more. Mechanisms underlying extreme survival are incompletely known, yet are important to improve prognosis and treatment. Extreme survival also provides an opportunity for cancer cells to evolve over decades. To evaluate the genomic characteristics of ELTS, we identified a cohort representative of the extreme tail of the metastatic survival curve and performed genomic analysis of blood and matched metastatic tumor. Methods: We identified a cohort of ELTS at our institution in an IRB-approved study. Eligible subjects had MBC and had lived a ≥10 years from the original breast cancer (BC) diagnosis with hormone receptor (HR)-positive disease or ≥5 years if HR-negative. Those with available archived tumor tissue were enrolled for genomic analyses. Archived samples were obtained from metastatic sites obtained late in the clinical course. Whole exome sequencing (WES) was performed on matched blood and tumor. Mutational profiles were qualitatively compared with publicly available reference somatic alterations in primary BC (Ctrl; cbioportal.org). Frequency of mutations is reported for known BC drivers. In an exploratory analysis to identify novel genes, the frequency of mutations in ELTS vs. Ctrl was compared for 15,659 well-covered genes. These were further evaluated for the ratio of non-synonymous to synonymous mutations, which is indicative of selective pressure. Results: 53 ELTS were identified; 13 had available archived tumor tissue and consented. The median survival from original BC diagnosis was 22 years, which corresponds to 99.8th percentile for survival, benchmarked to a cohort of MBC patients identified from the Surveillance, Epidemiology, and End Results-Medicare linked database. The 13 subjects had lived with metastatic disease for a median of 9 years (range 4-23). One subject had HR-/HER2+ BC and had lived 16 years with MBC. Three additional subjects had HR+/HER2+ BC with survival ranging from 19-25 years from original BC diagnosis. The remaining 9 subjects had HR+/HER2- BC with survival of 13-40 years from original diagnosis. Matched tumor/somatic WES achieved a mean tumor read depth of ˜600x and 96% coverage. Somatic mutations revealed near-expected rates of known driver alterations in commonly mutated genes such as PIK3CA, TP53, and CDH1 (Table). No mutations were found in PTEN or ESR1 in ELTS. Novel gene candidates were identified as having a high prevalence of mutations in this cohort including Androgen Receptor (10 of 13), MUC4 (12 of 13) MUC12 (10 of 13), and HRNR (12/13). Pathway analysis is ongoing. Conclusions: ETLS tumors obtained late in the clinical course have mutations in known cancer drivers near rates expected from primary BC. However, we have identified additional genes with an increased number of mutations either related to distinct genetic features of these cancers, or due to accumulated mutations that occur over the extended time for tumor evolution. Frequency of driver mutations in control and extreme long-long term survivorsGeneCtrl freqELTS freqGeneCtrl freqELTS freqPIK3CA0.350.38MAP2K40.020.38TP530.350.23NF10.040.15GATA30.120.08CBFB0.030.15CDH10.100.23ERBB20.030.08 Citation Format: Yan RE, Longhurst C, Lee K, Gilbert AD, Alexandridis RA, McGregor SM, Laffin JJ, Rocque G, Burkard ME. Whole exome analysis of extreme long-term survivors with metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-14.

Amino acid mutation in Plasmodium vivax dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes in Hormozgan Province, southern Iran.

Molecular analysis of antifolate resistance-associated genes-dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) of Plasmodium vivax is important in predicting the emergence of drug resistance to sulphadoxine-pyrimethamine (SP). The present study aimed to determine the polymorphism of dhfr and dhps genes in P. vivax field isolates. Samples from 80 microscopically diagnosed vivax malaria cases were collected from endemic areas of malaria in Hormozgan Province of Iran, from June 2010 to November 2015. The two sets of codons at position 33, 57, 58, 117, 173 of dhfr and 382, 383, and 553 of dhps genes were analysed by direct sequencing of PCR products. The majority of the isolates (70%) harboured a wild-type allele for P. vivax dhfr (Pvdhfr) and P. vivax dhps (Pvdhps). Mutations were detected in three codons of Pvdhfr (P33L, S58R and S117N) and single codon in Pvdhps (A383G). Novel mutations that have not been identified previously at codon 459 (D459A) of Pvdhps were also observed. The high prevalence of point mutation as well as the rising triple mutation of Pvdhfr and Pvdhps genotypes necessitate change in programmes and guidelines to eliminate P. vivax in future.

HIV Drug Resistance Mutations in Patients with HIV and HIV-TB Coinfection After Failure of First-Line Therapy: A Prevalence Study in a Resource-Limited Setting

Introduction:The present study aimed to report the prevalent HIV-1 drug-resistant mutations in patients with HIV-1 alone and tuberculosis (TB) coinfection alone to improve our understanding of the mutation patterns and aid treatment decisions.Methods:Patients with HIV-1 and HIV-TB on treatment for more than 1 year with suspected failure were recruited. Sequencing of protease and two-thirds of the region of reverse transcriptase gene was done for drug-resistant mutations.Results:In the HIV-TB group (n = 25), 88%, 92%, and 12% had mutations to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs), respectively. In the HIV-alone group (n = 25), 84%, 100%, and 4% had mutations to NRTIs, NNRTIs, and PIs, respectively. M184V, M41L, D67N, G190A, A98G, and K103N were the most common mutations seen.Conclusion:There is a high prevalence of drug-resistant mutations in HIV and HIV-TB coinfected patients.

Correlation between estrogen plasma level and miRNAs in muscle of Piedmontese cattle

A loss-of-function mutation of the myostatin gene has a very high prevalence in the Piedmontese cattle breed. The effect of such mutation is a double-muscle phenotype because of hypertrophy. However, differences in muscle mass development can still be detected in individuals of this breed. Such differences must be generated by other factors controlling skeletal muscle development. MicroRNAs are short noncoding RNA molecules that modulate gene expression at a post-transcriptional level. MicroRNAs have been demonstrated to be involved in skeletal muscle development, and some of them are controlled by steroid hormone signaling. Data on estrogen signaling are lacking, whereas more studies have been carried out on the effect of androgens. We aimed at identifying putative estrogen responsive miRNAs that might be involved in skeletal muscle development. At a slaughterhouse, we collected muscle samples from longissimus dorsi and blood samples. Blood 17β-estradiol concentration was assessed, and RNA was extracted from muscle samples. The animals we sampled were divided into groups according to estrogen blood concentration, and through qPCR expression, levels of 7 muscle-related miRNAs were evaluated. We found that miR-26b (P < 0.01), miR-27a-5p (P < 0.05), miR-27b (P < 0.05), and miR-199a-3p (P < 0.01) were differentially expressed among experimental groups. Expression levels of miR-26b were reduced approximately 50% in samples with a low blood estrogen concentrations, and the other miRNAs showed a tendency to increase their expression levels when blood estrogen levels were higher. The variations of the circulating concentrations of estrogen in Piedmontese cattle might influence muscle mass development through miRNAs and thus contribute to individual variability in a breed with a high prevalence of a myostatin point mutation.

Long Term AML Survivors Have Increased Mortality and High Prevalence of Clonal Hematopoiesis

Acute myeloid leukemia (AML) is a devastating disease; however 20%-30% of patients will achieve long term remission. The long-term follow up of AML patients who received bone marrow transplantation (BMT) is well documented. It remains unclear though whether AML long-term survivors who did not undergo BMT (LTSnoBMT) return to normal health. In order to answer this question a large cohort of AML patients and well matched controls are needed. For the present study, the electronic health records (EHR) of 4.2 million individuals covering over 16 years of follow up were studied. Morbidity and mortality of LTSnoBMT were compared to a large number of age-matched controls.We identified 177 LTS who were in remission two years after AML diagnosis of which 61 were LTSnoBMT. The mortality of LTSnoBMT 10 years after diagnosis was 20%-50% higher in comparison to controls in an age dependent manner with higher mortality among the elderly LTS (Figure 1A, B). While several lab results were different between the LTSnoBMT and controls, most strikingly the red cell distribution width (RDW) of the LTSnoBMT was much higher among elderly LTSnoBMT (Figure 1C). High RDW has been previously reported to correlate with mortality. Recent studies found that high RDW predict AML and even greater correlation of high RDW with mortality among individuals carrying age related clonal hematopoiesis (ARCH) mutations was detected.To investigate the genetic profile of LTSnoBMT, a deep-targeted sequencing method (covering 32 recurrently mutated genes in AML) was used. Remission samples (at least 2 years from diagnosis) from 28 LTSnoBMT were analyzed. In most cases mutations which were detected at diagnosis, were not present in the remission samples. ARCH defining events (most of them new) were found in 64% of the LTSnoBMT. To evaluate whether this high prevalence of ARCH related mutations among LTSnoBMT patients is related to chemotherapy exposure, we also studied 30 individuals with lymphoid malignancies who received chemotherapy and were in remission. The prevalence of ARCH related mutations in the controls was significantly lower (37%) than in the LTSnoBMT (Figure 2A) (p=0.035). Higher prevalence of IDH1/2 mutations was found among LTSnoBMT (Figure 2C) (p=0.017). Furthermore, 70% of the mutations detected in the LTSnoBMT were recurrent (i.e. has more than 5 occurrences in COSMIC data base) compared to 31% in the controls (Figure 2B) (p=0.001).Altogether, AML is a complex disease where even after the sustainable eradication of the malignant clone, the hematopoietic system/microenvironment does not normalize. The reported correlation between ARCH and mortality suggests that the high prevalence of ARCH related mutations among LTS might contribute to the high mortality that we observed. Our findings have both biological and clinical implications. Future studies should determine the reasons why AML - LTS have such high prevalence of ARCH with specific mutations and increased mortality. Furthermore, closer clinical follow up including mutation analysis should be considered for these individuals. [Display omitted] DisclosuresCilloni:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy. Ofran:Novartis: Other: Served on a Novartis advisory board.

High Prevalence of BTK Mutations on Ibrutinib Therapy after 3 Years of Treatment in a Real-Life Cohort of CLL Patients: A Study from the French Innovative Leukemia Organization (FILO) Group

High Prevalence of BTK Mutations on Ibrutinib Therapy after 3 Years of Treatment in a Real-Life Cohort of CLL Patients: A Study from the French Innovative Leukemia Organization (FILO) Group

Mosaic DNMT3A Germline Mutation As a Model for Mutant DNMT3A Competitive Advantage in the Blood Lineage

The DNA Methyltransferase 3A (DNMT3A) gene is recurrently mutated in a large spectrum of hematologic malignancies, including acute myeloid leukemia (AML). About 25% of adult AML patients carry mutations in DNMT3A and these mutations are generally associated with poor prognosis. DNMT3A mutations have been also associated with aged-related clonal hematopoiesis of indeterminate potential (CHIP). The high prevalence of DNMT3A somatic mutations in AML and CHIP implies that cells with mutated DNMT3A have a competitive advantage over wild-type (WT) cells, resulting in clonal expansion. However, the downstream molecular mechanisms that underlie this phenotype are not clear.Tatton-Brown-Raman syndrome (TBRS) is a rare genetic disorder caused by heterozygous germline mutations in DNMT3A, characterized by overgrowth and developmental delay. In one particular family, a group of 4 children out of 12 were diagnosed with TBRS and were found to be heterozygous carriers of DNMT3A-R771Q mutation (DNMT3AR771Q) inherited from their mosaic father. Thus, this individual provides a unique opportunity to study the long-term consequences of DNMT3A mutations, as he harbors both WT and mutant cells.From this mosaic individual, we generated lymphoblastoid cell lines (LCLs) from the peripheral blood (PB) and measured DNMT3AR771Q variant allele frequency (VAF) in the LCL pool as well as in PB, saliva and urine, all collected at the same time. Strikingly, DNMT3AR771Q VAF in the LCL pool and in PB was substantially higher than in saliva and urine (respectively 30%, 45%, 10%, 4%), implying that levels of DNMT3A mosaicism are tissue-specific and that cells with mutated DNMT3A tend to expand in the blood but not in epithelia (figure 1A and figure1B).One hypothesis for the prevalence of DNMT3A mutations in AML is that its loss reduces the effectiveness of DNA repair leading to increased mutational rates. In order to test this, we compared the mutational loads in individual LCL clones that were WT or DNMT3A mutant using whole genome sequencing. Surprisingly, no clear differences were observed between WT and DNMT3AR771Q mutant cells, indicating that clonal expansion is unlikely to be secondary to a general increase in mutational burden.To explore the impact of DNMT3AR771Q mutation on DNA methylation, we performed whole-genome bisulfite sequencing (WGBS) on two WT and two DNMT3AR771Q LCL clones. We identified ~31,500 differentially methylated regions (DMRs) between WT and mutant clones, with the majority of DMRs being hypomethylated. Hypomethylated DMRs were associated with gene regulatory regions, mainly promoters and enhancer regions. These data suggest that the DNMT3AR771Q mutation affects DNA methylation setting at genomic regions that can directly affect transcription.Canyons are large genomic regions of low methylation that often occur around master regulators such as homeobox-containing genes. We previously showed in mice that DNMT3A regulates DNA methylation at canyon edges, with loss of DNMT3A resulting in canyon expansion. In agreement, DNMT3AR771Q mutant clones displayed larger canyons, particularly at loci marked by H3K27Ac and H3K4me3 (figure 1C). Gene Ontology analysis of genes falling into expanded canyons showed a significant enrichment for leukemia and stem cell-related genes, including members of the HOX family. RNAseq analysis of DNMT3AR771Q mutant LCL clones confirmed the upregulation of key cancer-associated genes. These data suggest that DNMT3A mutations may promote clonal expansion through hypomethylation and overexpression of stem cell and cancer-related genesIn conclusion, by comparing WT and DNMT3Amutant LCL clones generated from the same individual, we show that DNMT3A mutations lead to significant hypomethylation and overexpression of key cancer-associated genes. Further studies on specific target genes will reveal critical pathways responsible for the clonal expansion of cells with mutated DNMT3A, paving the way for the development of new therapeutic strategies for malignancies with mutated DNMT3A. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Wnt1 is an Lrp5-independent bone-anabolic Wnt ligand.

WNT1 mutations in humans are associated with a new form of osteogenesis imperfecta and with early-onset osteoporosis, suggesting a key role of WNT1 in bone mass regulation. However, the general mode of action and the therapeutic potential of Wnt1 in clinically relevant situations such as aging remain to be established. Here, we report the high prevalence of heterozygous WNT1 mutations in patients with early-onset osteoporosis. We show that inactivation of Wnt1 in osteoblasts causes severe osteoporosis and spontaneous bone fractures in mice. In contrast, conditional Wnt1 expression in osteoblasts promoted rapid bone mass increase in developing young, adult, and aged mice by rapidly increasing osteoblast numbers and function. Contrary to current mechanistic models, loss of Lrp5, the co-receptor thought to transmit extracellular WNT signals during bone mass regulation, did not reduce the bone-anabolic effect of Wnt1, providing direct evidence that Wnt1 function does not require the LRP5 co-receptor. The identification of Wnt1 as a regulator of bone formation and remodeling provides the basis for development of Wnt1-targeting drugs for the treatment of osteoporosis.

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer

BackgroundPatients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC.MethodsWe again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth.ResultsCombination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2–3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation.ConclusionsCombination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

Antiretroviral resistance, genotypic characterization and origin of Human Immunodeficiency Virus among the infected wives of Intravenous drug users in Manipur

Increasing incidence of drug resistance is ascertained to be the main obstacles in limiting the virus among the human immunodeficiency virus (HIV) infected individuals. This study investigates the drug resistance mutations (DRMs), genetic variants and origin of transmitted drug resistance of HIV-1 among the HIV-1 infected wives of intravenous drug users (IDUs) in Manipur. 44 HIV pol gene sequences were generated from 56 blood samples by viral gene amplification and sequencing. Sequences were then analysed for drug resistance, genetic variants and origin. The result revealed that among the treatment naive cases, 35.7% had Transmitted Drug Resistance Mutations (TDRMs) while among treatment experienced cases, 50% had Acquired Drug Resistant Mutations (ADRMs). These TDRMs and ADRMs conferred resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and/or protease inhibitors (PIs). Majority of the isolated HIV-1 sequences (77.3%) were subtype C while 9.1% was discordant subtype, 6.8% was subtype B, 4.5% was CRF_01AE and 2.3% was URF_BC. TDRM strains were found to be introduced from Myanmar, Vietnam and mainland India. This study also reveals the appearance of CRF_01AE for the first time in Manipur. The finding of this study indicates high prevalence of drug resistant mutations and complex molecular epidemiology in Manipur.

Asymptomatic Natural Human Infections With the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi.

BackgroundIn Southeast Asia, Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously.MethodsMalariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR).ResultsAsymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20–40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection.ConclusionsAsymptomatic naturally acquired P. cynomolgi and P. knowlesi infections can both occur in humans.Clinical Trials RegistrationNCT01872702.

A13439 Cellular and Genetic Causes of Idiopathic Hyperaldosteronism

Objectives: Primary aldosteronism (PA) affects ∼10% of hypertensive patients and has unilateral and bilateral forms (30%:70%). Most unilateral PA is caused by computed tomography (CT)- detectable aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase), and frequently harbor somatic mutations in aldosterone-regulating genes (KCNJ5 >> CACNA1D). The etiology of the more common bilateral PA (often termed idiopathic hyperaldosteronism [IHA]) has long been believed to result from diffuse hyperplasia of adrenal cortex cells without incorporation of CYP11B2 expression. Methods: Herein, fifteen IHA adrenals from a multi-institution cohort were examined using CYP11B2 immunohistochemistry and next generation sequencing (NGS). Results: CYP11B2 immunoreactivity in the adrenal cortex was diffusely positive in only 4/15 IHA adrenals, arguing against this as the common cause of IHA. Instead, all adrenals harbored at least one CT-undetectable CYP11B2-expressing cell clusters (termed aldosterone-producing microscopic lesions [APML]), which existed sporadically in adrenal cortex. The median number per case and individual area of APML in IHA was significantly larger than those in normotensive controls. NGS of DNA from 99 adrenal APML demonstrated somatic mutations in CACNA1D (57 / 58%) and KCNJ5 (1 / 1%). Conclusion: These data indicate that the most common cause of PA, IHA, results from the accumulation of image-undetectable aldosterone producing cells harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in CACNA1D, an L-type calcium channel, indicate a potential actionable therapeutic target to inhibit aldosterone overproduction in IHA patients.

P1.15-26 A Review of Colombian National Administrative Cancer Registry (NACR) Data to Evaluate Healthcare Delivery and Biologics Use

The Office of High Cost of the Colombian Health Ministry created the National Administrative Cancer Registry (NACR) data first in 2015 to provide comprehensive cancer data to improve cancer outcomes while serving as a model for other resource-limited countries. Despite new targeted therapies throughout the world, the benefits of these therapies have not reciprocated in lower resource settings, notably in Latin America. The purpose of this study is to investigate aspects of the NACR data that underscore some of the health care limitations of lung cancer treatment in Colombia. We obtained National Administrative Cancer Registry (NACR) data from the High-Cost Diseases Office (Cuenta de Alto Costo [CAC]) collected in 2015 and released in 2016. All cancer cases diagnosed in the country are reported by payers and providers otherwise there are no payments for services rendered, assuring that the registry is representative. We use descriptive statistics for presentation of data and comparisons. A total of 3,082 patients were analyzed of which 2,043 (66.29%) had contributive insurance, 820 (26.60%) had subsidized insurance, and 98 (3.18%) had special or exempt insurance. Four patients (0.12%) had no insurance. Of newly diagnosed patients, the median number of days from suspicion to diagnosis was 27 days (IQR = 12-45 days, n = 491) with the predominant range of patients with contributive insurance being 30-59 days, and for subsidized insurance being 15-29 days. The median number of days from diagnosis to first treatment was 31 days (IQR=14-62, n=346) with the predominant range for patients with both contributive and subsidized insurance being 30-59 days. There was a greater percentage of Stage IV cancers in patients with subsidized (34%) than contributive (23%) insurance. Of those receiving chemotherapy (n=275), 52% received carboplatin, 28.3% received pemetrexed, 27.6% received cisplatin, 25.4% received paclitaxel, 10.9% received bevacizumab, and 5.1% received erlotinib; no patients received nivolumab or pembrolizumab. Based on findings from NACR, the wait time from suspicion to treatment took nearly two months underscoring the need for better streamline of lung cancer care. Also, data shows a low percentage of use of newer therapies, including EGFR-targeted agents despite a high prevalence of mutations, which are present in around a quarter of patients in Colombia (Raez, 2017). Colombia can strongly benefit from increased access to molecular testing and biologics given the future direction of lung cancer therapy.

Cellular and Genetic Causes of Idiopathic Hyperaldosteronism.

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.

High frequency of drug resistance mutations in the HBV genome in ART-experienced HIV-coinfected patients in southwestern Nigeria.

HBV and HIV infections are highly endemic in sub-Saharan Africa and Nigeria while HBV-HIV coinfection is not uncommon. Antiretroviral (ART)-treatment for HIV can affect HBV whereby antiviral resistance mutations in the HBV genome can be selected. Here, we determined the prevalence of resistance mutations among ART-experienced and ART-naive HIV-HBV-coinfected patients in southwestern Nigeria. A total of 81 serum samples from HBV-HIV-coinfected patients who were either ART-naive or received lamivudine (3TC)-containing ART-therapy and HBV-monoinfected patients were analysed. Hepatitis B surface antigen (HBsAg) was detected using ELISA. HBV-positive samples were confirmed by PCR amplification of the surface and polymerase regions. Mutations conferring drug resistance to HBV were analysed by direct sequencing. Phylogenetic analysis was performed to identify the HBV genotype. Of the 81 HBsAg-positive samples, 27 had detectable HBV DNA by real-time PCR with mean viral loads of 6.77 log IU/ml. Phylogenetic analyses showed a predominance of HBV genotype E. A high prevalence (22.2%; 6/27) of HBV resistance mutations among ART-experienced HBV-HIV-coinfected patients was detected. However, a relatively high selection rate of resistance mutations in drug-naive HIV-HBV-coinfected (3.7%; 1/27) and in HBV-monoinfected patients, potential drug resistance mutations (7.4%; 2/27) were also observed. HBV polymerase amino acid substitutions found included rtV173L, rtL180M, rtM204V, rtK212R, rtS213T, rtV214A, rtL229V and rtP237A/S. Drug resistant mutations were detected frequently in ART-experienced HIV-HBV patients. Well-coordinated antiviral therapy for HIV patients coinfected with HBV should include proper HBV diagnosis and resistance testing to minimize the emergence and spread of antiviral drug resistance.

Prevalence and Spectrum of Breast Cancer Inherited Mutations in Uganda and Cameroon Women

Background: Breast cancer among indigenous Africans is characterized by higher prevalence of triple-negative disease and poor prognosis. A previous study in Nigeria reported a strikingly high prevalence of deleterious germline mutations in BRCA1 and BRCA2 among Nigerian women with breast cancer. It is unknown if this is true in other sub-Saharan African countries. Aim: The objective of this study is to determine the frequency of germline mutations among an unselected sample of women in Africa. Methods: We conducted a case-control study of breast cancer in Uganda and Cameroon to investigate genetic and nongenetic risk factors for breast cancer. Breast cancer cases were enrolled in two tertiary hospitals in the two countries, unselected for age at diagnosis and family history. Controls who were free of breast cancer were enrolled in the same hospitals and matched to cases on age. A 24-gene sequencing panel was used to test germline mutations in cases and controls. Results: There were 176 cases and 177 controls with a mean age at diagnosis of 46.2 years for cases and mean age at interview of 46.7 years for controls. Among cases, 18.2% carried a pathogenic mutation in a breast cancer gene: 6.3% in BRCA1, 6.3% in BRCA2, 1.7% in ATM, 1.1% in PALB2, 0.6% in BARD1, 0.6% in CDH1, 0.6% in TP53, and 1.2% in any of 17 other genes. Among controls, 2.3% carried a pathogenic mutation in one of the 24 susceptibility genes. Cases were 9.6-fold more likely to carry a mutation compared with controls (odds ratio=9.61, 95% confidence interval: 3.28-38.1; P &lt; 0.001). The mean age of breast cancer cases with pathogenic BRCA1 mutations was 38.3 years compared with 46.7 years among other cases without such mutations ( P = 0.03). There was a trend that cases with a positive family history had higher chance of carrying a mutation (33.3%) than cases without (17.1%), but few cases reported a positive family history. Conclusion: Our findings confirm the earlier report of a high proportion of deleterious mutations in BRCA1 and BRCA2 among breast cancer patients in sub-Saharan Africa. As most of these women present with advanced breast cancer, there is an urgent need to improve access to genomic testing and life saving cancer medicines including chemotherapy and clinical trials of novel agents like PARP inhibitors. Given the high burden of inherited breast cancer, genetic risk assessment should be integrated into cancer control plans in sub-Saharan Africa.

157P - Short-term responders of non-small cell lung cancer patients to EGFR tyrosine kinase inhibitors display high prevalence of TP53 mutations and primary resistance mechanisms

157P - Short-term responders of non-small cell lung cancer patients to EGFR tyrosine kinase inhibitors display high prevalence of TP53 mutations and primary resistance mechanisms

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma

AbstractMature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

Short-Term Responders of Non–Small Cell Lung Cancer Patients to EGFR Tyrosine Kinase Inhibitors Display High Prevalence of TP53 Mutations and Primary Resistance Mechanisms

Non–small cell lung cancer (NSCLC) with activating EGFR mutations in exon 19 and 21 typically responds to EGFR tyrosine kinase inhibitors (TKI); however, for some patients, responses last only a few months. The underlying mechanisms of such short responses have not been fully elucidated. Here, we sequenced the genomes of 16 short-term responders (SR) that had progression-free survival (PFS) of less than 6 months on the first-generation EGFR TKI and compared them to 12 long-term responders (LR) that had more than 24 months of PFS. All patients were diagnosed with advanced lung adenocarcinoma and harbored EGFR 19del or L858R mutations before treatment. Paired tumor samples collected before treatment and after relapse (or at the last follow-up) were subjected to targeted next-generation sequencing of 416 cancer-related genes. SR patients were significantly younger than LR patients (P < .001). Collectively, 88% of SR patients had TP53 variations compared to 13% of LR patients (P < .001). Additionally, 37.5% of SR patients carried EGFR amplifications compared to 8% of LR patients. Other potential primary resistance factors were also identified in the pretreatment samples of 12 SR patients (75%), including PTEN loss; BIM deletion polymorphism; and amplifications of EGFR, ERBB2, MET, HRAS, and AKT2. Comparatively, only three LR patients (25%) were detected with EGFR or AKT1 amplifications that could possibly exert resistance. The diverse preexisting resistance mechanisms in SR patients revealed the complexity of defining treatment strategies even for EGFR-sensitive mutations.

High prevalence of virological failure and HIV drug mutations in a first-line cohort of Malawian children.

Drug resistance mutations (DRMs) increasingly jeopardize paediatric HIV programmes in sub-Saharan Africa. As individual monitoring of DRMs and viral loads has limited availability, population data on DRMs are essential to determine first-line susceptibility. Paediatric data from sub-Saharan Africa are scarce and unavailable for Malawi. To determine the prevalence of virological failure (VF) and DRMs among ART-naive HIV-infected Malawian children during the first year of first-line ART. In a prospective cohort of HIV-infected Malawian children, on first-line treatment, children were followed monthly; blood was collected for viral load testing (6 and 12 months) and genotypic resistance testing (12 months). VF was defined as at least one viral load >1000 copies/mL or death after 6 months of ART. DRMs were identified and susceptibility to NRTIs and NNRTIs was scored using the Stanford algorithm and by calculating genotypic susceptibility scores (GSSs). VF occurred in 66% (23/35) of the children during 12 months of follow-up. DRMs were detected in 44% (15/34); all had NNRTI resistance and 12% (4/34) had dual-class NNRTI/NRTI resistance. Reduced susceptibility (DRMs and GSS <3) was seen in 41% (14/34) to their current first-line regimen. High-level resistance was most common for nevirapine [26% (9/34)]. In this first report on VF and DRMs in children on first-line ART in Malawi, the rates of VF and DRMs were alarmingly high. Paediatric HIV programmes in sub-Saharan Africa should emphasize programmatic evaluation of VF and include detection of DRMs to adjust and design adequate first- and second-line regimens and prevent widespread resistance in children.