SDHB Research Articles

Baseline Sensitivity of Botrytis cinerea Isolates from Strawberry to Isofetamid Compared to other SDHIs.

Succinate dehydrogenase inhibitors (SDHIs) are the fungicides most commonly used to control Botrytis fruit rot on commercial strawberry in Florida. The medium-to-high risk of selection of resistance in the causal agent Botrytis cinerea is a threat to the efficacy of this fungicide group. In this study, we characterized the sensitivity of B. cinerea to the SDHI isofetamid, evaluated the SdhB gene mutation associated with resistance, and monitored resistance frequencies to five SDHI fungicides for two consecutive seasons. EC50 values of 70 isolates were obtained using the spiral gradient dilution (SGD) method and averaged 0.098 µg/ml of isofetamid. EC50 averages of 3.04 and >500.00 µg/ml were obtained for isolates with the N230I and P225F mutations indicating moderate and high resistance to isofetamid, respectively. A total of 565 B. cinerea isolates collected during 2015-2016 and 2016-2017 seasons from strawberry nurseries and Florida production fields were evaluated using conidial germination assays. Results for the first season showed resistance frequencies of 95, 33, 21, 25, and 0% to boscalid, penthiopyrad, fluopyram, benzovindiflupyr, and isofetamid, respectively. The respective resistance frequencies for the following season were 91, 95, 44, 27, and 1.3%. Only three isolates were found to be moderately resistant to isofetamid during the second season, and the mutation N230I was identified after sequence analysis. These isolates were confirmed to be resistant to isofetamid in fruit assays with disease incidence of 55.6 to 77.0%; however, the conidial production of the isolates was inhibited by an average of 83.9%. In general, isofetamid efficacy was higher than the other evaluated SDHIs, but a slight increase in resistance frequencies was observed in our study.

Analysis of testis metabolome and transcriptome from the oriental river prawn (Macrobrachium nipponense) in response to different temperatures and illumination times

A better understanding of the mechanisms underlying the male sexual differentiation of Macrobrachium nipponense is urgently needed in order to maintain sustainable development of the M. nipponense industry. Environmental factors, especially temperature and illumination, have dramatic effects on gonadal development. The aim of the present study was to identify key genes and metabolites involved in the male sexual differentiation and development of M. nipponense through integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times. A total of 268 differentially abundant metabolites and 11,832 differentially expressed genes (DEGs) were identified. According to integrated metabolomics and transcriptome analyses, glycerophospholipid and sphingolipid metabolism was predicted to have dramatic effects on the male sexual differentiation and development of M. nipponense. According to the KEGG enrichment analysis of DEGs, oxidative phosphorylation, glycolysis/gluconeogenesis, the HIF-1 signaling pathway, the citrate cycle, steroid hormone synthesis, and the spliceosome complex were predicted to promote male differentiation and development by providing adenosine triphosphate, promoting the synthesis of steroid hormones, and providing correct gene products. Quantitative polymerase chain reaction analysis and in situ hybridization showed that the SDHB, PDE1, HSDL1, CYP81F2, SRSF, and SNRNP40 genes were differentially expressed, suggesting roles in the male sexual differentiation and development of M. nipponense. Strong candidate sex-related metabolic pathways and genes in M. nipponense were identified by integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times, as confirmed by PCR analysis and in situ hybridization.

Effects of anti–TNF-alpha therapy on hemoglobin levels and anemia in patients with inflammatory bowel disease

BackgroundTumor necrosis factor-α (TNF-α) is involved in inducing inflammatory anemia. The potential effect of anti–TNF-α agents on anemia in inflammatory bowel diseases (IBD) is still unknown. MethodsAnalytical data and disease characteristics from 362 IBD patients [271 CD/91UC) treated with anti-TNF-α drugs were retrospectively collected. Effects on disease activity, blood markers and prevalence of anemia were assessed after 6 and 12 months of therapy. Results29.3% patients presented anemia at baseline, and significantly reduced to 14.4% and 7.8% after 6 and 12 months of therapy, respectively. Mean ± SD Hb levels increased significantly at month 6, and this increase was sustained at 12 months. Serum markers of iron metabolism increased significantly compared to baseline, as disease activity measured by C-reactive protein (CRP) was reduced. All these effects were observed independently for CD and UC, and were independent of iron supplementation during treatment. Anemia at baseline (OR 4.09; 95%CI 1.98–8.45) and elevated CRP (OR 3.45; 95CI 1.29–9.22) were independently associated with risk of persistent anemia, as well as iron replacement during therapy (OR 4.36; 95%CI 2.07–9.16). ConclusionsControlling disease activity with anti-TNF- α therapy significantly and independently associated with resolution of anemia in IBD, with no relevant role for iron replacement therapy.

Pediatric gastrointestinal stromal tumor: Report of two novel patients harboring germline variants in SDHB and SDHC genes

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and rarely occur in pediatric patients. 85% of pediatric GISTs and 15% of adult GISTs lack of KIT or PDGFRA mutations. 40% of these “wild-type” GISTs present loss of function mutations in genes encoding for the subunits of the succinate dehydrogenase (SDH) complex. Germline mutations in SDH complex genes have been described in patients with the Carney–Stratakis syndrome (CSS), a rare inherited condition that predisposes to GIST and paraganglioma. We report two pediatric patients with multifocal GIST, harboring respectively a novel and a previously reported loss-of-function germline variant, in SDHC and SDHB genes.

Loss of BAP1 in Pheochromocytomas and Paragangliomas Seems Unrelated to Genetic Mutations.

Breast cancer-associated protein 1 (BAP1) gene is a broad-spectrum tumor suppressor. Indeed, its loss of expression, due to biallelic inactivating mutations or deletions, has been described in several types of tumors including melanoma, malignant mesothelioma, renal cell carcinoma, and others. There are so far only two reports of BAP1-mutated paraganglioma, suggesting the possible involvement of this gene in paraganglioma (PGL) and pheochromocytoma (PCC) pathogenesis. We assessed BAP1 expression by immunohistochemistry (IHC) in a cohort of 56 PCC/PGL patients (and corresponding metastases, when available). Confirmatory Sanger sequencing (exons 1-17) of BAP1 has been performed in those samples which resulted negative by IHC. BAP1 nuclear expression was lost in 2/22 (9.1%) PGLs and in 12/34 (35.3%) PCCs, five of which harboring a germline mutation predisposing the development of such tumors (MENIN, MAX, SDHB, SDHD, and RET gene). Confirmatory Sanger sequencing revealed the wild-type BAP1 status of all the analyzed samples. No heterogeneity between primary and metastatic tissue was observed. This study documents that the loss of BAP1 nuclear expression is quite a frequent finding in PCC/PGL, suggesting a possible role of BAP1 in the pathogenesis of these tumors. Gene mutations do not seem to be involved in this loss of expression, at least in most cases. Other genetic and epigenetic mechanisms need to be further investigated.

2039P - Driving solo? Investigation into collaborating mutations in SDH-deficient neoplasia

BackgroundSuccinate dehydrogenase (SDH) is a highly conserved component of energy production and metabolism. Genetic or epigenetic inactivation of SDH (dSDH) creates a state of intracellular pseudohypoxia that is uniquely oncogenic for subtypes of GIST, renal cell carcinoma (RCC), paraganglioma/pheochromocytoma (PGL/Pheo), and pituitary adenoma/carcinoma (PAC). dSHD stems from homozygous loss of one of four subunit encoding genes SDHA, SDHB, SDHC, or SDHD (aka, SDHx). dSDH is not targetable, and preclinical models are poorly viable. In the current study we analyze comprehensive genomic profiles (CGP) from a dSDH pan-tumor cohort for potential therapy-enabling non-SDHx genomic alterations and biomarkers (GA). Methods231,706 clinical grade CGP were searched for dSDH GIST, RCC, PGL/Pheo, and PAC. GA were determined on 1.1Mb genome sequence encompassing up to 324 cancer genes. dSDH was further established by histopathology review and analysis of SDHx homozygous loss. Germline and zygosity status of SDHx variants were performed by SGZ algorithm. Non-SDHx GA were assessed for therapeutic actionability. Results82 SDH-deficient neoplasms were analyzed (Table). 0 of 38 GIST had highly actionable GA. In one case a subclonal KIT variant was identified in a recurrence of an SDHA-mutant GIST treated with Imatinib for several years. No clonal canonical driver kinase mutations were identified in KIT, PDGFRA, NF1, BRAF, FGFR1 or NTRK1-3. 0 of 36 dSDH PGL/Pheo and 0 of 1 PAC had potential therapeutic options. 2 of 7 dSDH RCC reported potential treatment options for oncogenic variants in EGFR and CDK4. No dSDH tumors had high TMB, microsatellite instability, or LOH score indicative of homologous recombination defect (HRD). Overall, 2 of 82 tumors (2.4%) harbored highly actionable GA.Table2039PTable82 SDH-deficient tumorsGIST (n=38)PGL/Pheo (n=36)RCC (n=7)PAC (n=1)Age (mean)33404944Sex ratio (M/F)16/2124/124/30/1SDHA111231SDHB72130SDHC4110SDHD2200SDHx-WT14000%SDHx germline696410050GA/tumor (pathogenic non-SDHx)0.580.892.290MSI0000TMB (mut/Mb, mean)1.862.430.9HRD (LOHscore >16%)0000 ConclusionsThis study identifies SDH deficiency as a lone driver of malignancy and without associated actionable GA in>97% of cases, and underscores the need for novel therapeutic approaches targeting SDH deficiency itself. Legal entity responsible for the studyFoundation Medicine. FundingFoundation Medicine. DisclosureJ.K. Killian: Full / Part-time employment: Foundation Medicine. D.C. Pavlick: Full / Part-time employment: Foundation Medicine. E.S. Sokol: Full / Part-time employment: Foundation Medicine. M. Montesion: Full / Part-time employment: Foundation Medicine. D.X. Jin: Full / Part-time employment: Foundation Medicine. B. Kaplan: Full / Part-time employment: Foundation Medicine. D. Lin: Full / Part-time employment: Foundation Medicine. J. Vergilio: Full / Part-time employment: Foundation Medicine. J.A. Elvin: Full / Part-time employment: Foundation Medicine. N. Ngo: Full / Part-time employment: Foundation Medicine. E. Severson: Full / Part-time employment: Foundation Medicine. S. Ramkissoon: Full / Part-time employment: Foundation Medicine. D. Duncan: Full / Part-time employment: Foundation Medicine. C. Edgerly: Full / Part-time employment: Foundation Medicine. A. Hemmerich: Full / Part-time employment: Foundation Medicine. G.M. Frampton: Full / Part-time employment: Foundation Medicine. V.A. Miller: Full / Part-time employment: Foundation Medicine. S.M. Ali: Full / Part-time employment: Foundation Medicine. J.S. Ross: Full / Part-time employment: Foundation Medicine. All other authors have declared no conflicts of interest.

145P - Breast cancer in young women of Kazakh population depending on germline mutations: Results of next-generation sequencing

BackgroundBreast cancer (BC) represents the most common malignancy and has the highest mortality among women, both in the world and in Kazakhstan. Approximately 20-30% of cases of hereditary breast cancer are caused by presence of BRCA1 and BRCA2 genes defects. Also, there are additional genes which can increase the risk of BC and they are still under study.The aim of this study was to identify new, detectable and objective markers of key cancer genes by next-generation sequencing (NGS) in BC patients. MethodsThe study included 194 unrelated patients (the av/age 34.25 ± 4.56) with BC. Genomic DNA was obtained from peripheral blood,next-generation sequencing was performed using TruSightCancer Kit on the MiSeq platform,studio Variant was used to annotate genetic variants. ResultsIn total, 61 pathogenic variants (all in heterozygous state) were found in 56 (28.9%) patients,the vast majority of variants located in BRCA1 (n=19/56; 33.9%), 15 in BRCA2 (26.8%). The frequency of pathogenic variants in genes TP53 (8.9%), PALB2 (5.4%), MSH6 (5.4%), CHEK2 (3.6%), SDHB (3.6%), and WRN (3.6%) were higher than those genes APC, ATM, FANCA, FANCM, MSH2, NBN, NF1, PMS1, PMS2, and XPA which include only one deleterious variants. The analysis of mutation type has revealed 28 frameshift mutations, 15 stop-gain mutations, 8 missense mutations, 8 splice site variants, 1 start lost variant, and 1 synonymous variant. In total, 43 mutations were unique, 15 of them represented novel variants. Those new mutations have not been previously mentioned in the LOVD and ClinVar databases and have not been described in publications. Population frequency of all detected pathogenic mutations in 1000G, ESP6500 and ExAC databases were less than 1%. 73.8% of the mutations were available in the dbSNP database. The most common pathogenic variants were c.5329dupC and c.5341-2delA (c.5278-2delA) in BRCA1 gene, accounting for 10.7% (n=6/56) and 8.9% of patients (n=5/56), respectively. ConclusionsNGS showed frequent and novel germline mutations in BRCA1/2, CHEK2, TP53 and PALB2. After the final statistical data processing, diagnostic and prevention tools for key genes will be developed and included in the National guidelines for cancer diseases. Legal entity responsible for the studyKazakh Institute of Oncology and Radiology Institute of General Genetics and Cytology. FundingThe Ministry of Healthcare of the Republic of Kazakhstan. DisclosureAll authors have declared no conflicts of interest.

Abstract 2937: Preclinical evaluation of new therapeutic strategies on SDHB invalidated clones from human pheochromocytoma cells

Abstract Introduction: Malignant paraganglioma/pheochromocytoma (MPP) are very rare neuroendocrine tumors with heterogeneous prognostic and no gold-standard treatment. MPP can be associated with germline mutations at SDHB gene which encode for a TCA enzyme, the succinate dehydrogenase that catalyzes the oxidation of succinate to fumarate. When mutated, SDHB losses its function, leading to succinate accumulation by inhibiting 2-oxoglutarate dependent dioxygenases involved in methylation and angiogenesis. Thus alkylating agents, antiagniogenic and demethylating drugs are potential strategies for MPP patients. Aim: This project aimed to establish preclinical models of SDHB invalidated human cell line and to characterize new therapeutic strategies. Material & Methods: Invalidation of SDHB gene has been performed using the CRISPR-Cas9 technology on a human pheochromocytoma cell line (hPHEO1). Isogenic clones have been characterized at the genetic, cellular, proteic, and enzymatic levels through sequencing, immunohistochemistry, western-blotting and enzymatic assays, respectively. The antiproliferative effects of three drugs (temozolomide, sunitinib, azacytidine) have been explored and half maximal inhibitory concentrations (IC50) have been determined on the parental cell line and the 4 clones using the WST1® assay. Results: Four heterozygote isogenic clones have been obtained with 3 different genetic alterations (deletion-insertion). Expression of Ki67 was increased in the SDHB+/- clones. SDHB expression was partially reduced in clones 1 and 2 and almost totally suppressed in clones 3 and 4. For temozolomide and sunitinib, IC50 were significantly lower in SDHB+/- clones (~140 µM for temozolomide and ~1.4 µM for sunitinib) compared to the parental cell line (~1300 µM for temozolomide and ~6 µM for sunitinib) whereas IC50 were similar for azacytidine (~3.5 µM). No significant difference was observed between clones. Conclusion: In this study we obtained 4 SDHB invalidated isogenic clones from the human hPHEO1 cell line, pretty useful for preclinical evaluations in this rare disease. In agreement with literature clones were more sensitive to temozolomide but also to sunitinib. Metabolomic characterization of hPHEO1 and SDHB+/- clones is currently under investigation at baseline and after IC50 treatment of the 3 therapeutic strategies. Drugs’ combinations will be studied in a further step. Citation Format: Constance Lamy, Julien Hadoux, Sylvere Durand, Abir Alghuzlan, Julie Riviere, Deborah Lefevre, Amaury Bongers, Jean-Yves Scoazec, Eric Baudin, Angelo Paci, Sophie Broutin. Preclinical evaluation of new therapeutic strategies on SDHB invalidated clones from human pheochromocytoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2937.

Preimplantation genetic diagnosis for a single gene mutation for succinate dehydrogenase subunit B (the genetic basis for malignant paraganglioma) with successful pregnancy

Purpose: To report the first successful case of the birth of normal baby using preimplantation diagnosis for hereditary paraganglioma (PGL) followed by embryo transfer and to emphasize the importance of using mild follicle stimulating hormone (FSH) stimulation for IVF in women with diminished oocyte reserve. Materials and Methods: The female partner had a baseline serum FSH of 16.5 mIU/mL and the male partner had the malignant hereditary form of the PGL - pheochromocytoma (PGL/PCC) syndrome related to a mutation of the nuclear mitochondrial enzyme, succinate-dehydrogenase enzyme (specifically the B subunit) (SDHB). He was suffering from the malignant transformation of these neuroendocrine tumors. Before his death, he wanted to enjoy for a short time a baby with his own genes but feared passing the SDHB autosomal gene mutation to their child. Results: Eight blastomeres from eight embryos biopsied on day 3 were tested for SDHB gene by polymerase chain reaction. These eight metaphase II oocytes were obtained following mild FSH stimulation (150 units from day 3). Intracytoplasmic sperm injection (ICSI) was performed using the frozen/thawed sperm of this 35-year-old male suffering from a malignant pheochromocytoma. Five of the eight embryos were found to have mutations of the SDHB gene. Two embryos with normal SDHB were transferred and one resulted in a healthy baby. Genetic testing of the baby confirmed the absence of the SDHB mutation. Discussion: The successful pregnancy supports, but does not prove, the importance of using mild FSH stimulation in the presence of diminished oocyte reserve to inhibit the iatrogenic development of a much higher percentage of embryos with aneuploidy.

Genetic and epigenetic alterations of SDH genes in patients with sporadic succinate dehydrogenase-deficient gastrointestinal stromal tumors.

This study aimed to investigate the association of SDH gene mutations and promoter methylation with succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs) and to further discuss the potential molecular mechanisms underlying SDHB expression loss in these tumors. First, a total of 26 patients with SDH-deficient GISTs were selected by identifying the loss of SDHB protein expression and wild-type for KIT and PDGFRa mutations. Then SDH gene mutations and promoter methylation were detected by DNA sequencing and methylation-specific polymerase chain reaction, respectively, and the clinical and pathological data of SDH-deficient GISTs patients were collected and analyzed accordingly. The results of genetic testing demonstrated that 38.46% (10/26) of these patients harbored mutations in SDHB, SDHC, and SDHD genes (3 cases with double mutations). Besides, aberrant promoter methylation of SDH genes was detected in 10 out of 26 cases (38.46%), including 8 cases in SDHA gene, 3 cases in SDHB gene, 1 case in both SDHA and SDHB genes. It is suggested that SDH gene mutations and promoter methylation may contribute to the loss of SDH protein expression in sporadic SDH-deficient GISTs. This study indicated that the genetic and epigenetic alterations of SDH genes may occur during tumor formation.

Positive/retained SDHB immunostaining in renal cell carcinomas associated to germline SDHB-deficiency: case report

BackgroundAccording to WHO, succinate dehydrogenase (SDH)–deficient renal cell carcinoma is characterized by negative immunostaining for SDHB, which remains positive in non-tumor tissue despite germline mutations in the SDHB gene. We now report a patient with a SDHB mutation, c.166_170del (p.Pro56Tyrfs*5) who developed renal cell carcinomas with characteristic morphological features of SDH-deficient renal cell carcinoma but had positive SDHB immunostaining.Case presentationWithin a 6-year period, the patient developed two different renal cell carcinomas, which had characteristic morphological features of SDH-deficient renal cell carcinoma (uniform cells characteristically displaying eosinophilic granular material intermixed with fewer cells exhibiting clear intracytoplasmic inclusions and bland centered nuclei) but displayed immunohistochemistry for SDHB with a cytoplasmic granular positivity (mitochondrial pattern) in tumor cells. For the second case, this was initially interpreted as positive by IHC, but on review some subtle differences were identified.ConclusionsSDHB immunostaining may be positive in renal cell carcinoma associated to germline SDHB deficiency which have other typical morphological features. Immunohistochemistry interpretation may be complex.

Boscalid Resistance in Blumeriella jaapii: Distribution, Effect on Field Efficacy, and Molecular Characterization.

Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of tart cherry (Prunus cerasus L.) trees, leading to early defoliation that results in uneven ripening and poor fruit quality in the current season, reduced fruit set in the following season, and increased potential for winter injury and tree death. Pristine (BASF Corporation, Research Triangle Park, NC), a commonly used fungicide for CLS management in Michigan, is a premix of boscalid, a succinate dehydrogenase inhibitor, and pyraclostrobin, a quinone outside inhibitor. Reduced efficacy of Pristine for CLS control was observed in field trials and commercial orchards and highlights the importance of fungicide resistance monitoring. A total of 1,189 isolates from 31 commercial orchards in Michigan, 111 isolates from nontreated trees (four locations in Michigan and two locations in Ohio), and 133 isolates from a research orchard were collected during 2010, 2011, and 2012 and assayed on boscalid-amended media at concentrations ranging from 0 to 25 μg ml-1. Because of the very slow growth rate of B. jaapii in culture, we determined the minimum inhibitory concentration (MIC) of boscalid as opposed to the effective concentration that inhibits mycelial growth to 50% of the control. Isolates from nontreated trees had MIC values ranging from 0.1 to 0.5 μg ml-1; the MIC of isolates from commercial orchards ranged from 0.1 to >25 μg ml-1, and isolates from the research orchard ranged from 2.5 to >25 μg ml-1. Isolates with MIC values ≥25 μg ml-1 were considered boscalid resistant and comprised 0% of the nontreated isolates, 30.4% of the commercial isolates, and 42.1% of the research orchard isolates. Sequencing of the sdhB gene of resistant isolates led to the detection of the amino acid mutation H260R, which is known to confer boscalid resistance in other phytopathogenic fungi. Our results indicate that the occurrence of the H260R mutation in Michigan populations of B. jaapii is correlated with the reduction in sensitivity to boscalid observed in commercial orchards.

SAT-319 Long-term Follow-up of Two Unrelated Boys With SDHB-Related Malignant Paragangliomas

Background: Paragangliomas (PGLs) are rare neuroendocrine cell tumors and due to its rarity, there is no current standardized treatment and optimal long-term follow-up for metastatic disease. We report two cases of SDHB-related malignant PGL in boys with more than 5 years of follow-up. Clinical case: Patient 1 (P1) presented at the age of 13 with hypertension crisis, after a history of paroxystic tachycardia and sweating since 9. Fractionated 24-hour urinary metanephrines showed high Normetanephrine (NMT). CT showed bilateral lung lesions, the largest in right basis (6.1x5.3cm) in close contact with the 7/8th ribs and vertebrae, enlarged thoracic lymph nodes, and a 2.2 cm tumor near the left carotid. The patient went through a debulking surgery of the tumors. Patient 2 (P2) presented with abdominal pain at the age of 7. CT showed an abdominal tumor that was surgically resected. In both cases immunohistochemistry was compatible with PGL. P2 tumor was a non-secreting PGL, with high plasma chromogranin A prior to surgery. Both subsequently developed multiple bone metastases, and in case 2, additional recurrent retroperitoneal metastases. Adjuvant therapeutic 131I-MIBG, until a total dose of 900mCi (P1) and 950mCi (P2,) was performed. Over a 5- and 7-year follow-up, respectively, sustained stable disease has been documented, with control of tumor burden, despite the presence of several metastatic foci, mostly in bones. P1 has maintained mild hypertension controlled with diltiazem 30mg bid and has been annually tested for urinary NMT, which is decreasing though still mildly elevated, plus imaging (MRI and/or PET-CT). Case 2 has been clinically asymptomatic with six-monthly chromogranin A determinations currently in the normal range, and annual imaging (MIBG scintigraphy, MRI and/or PET-CT). As both cases have bone metastatic disease, 5mg intravenous zoledronic acid was recently started. Sequencing of the coding exons and exon-intron boundaries of the SDHB gene found no mutations in P1, whereas a heterozygous frameshift deletion in exon 4 was found in P2 (c393delA). In P1, complementary Multiple Ligand-Binding Probe Amplification revealed a heterozygous deletion of SDHB exon 1. P1’s mother genetic screening was negative and the father refused testing. Six P2’s relatives were found to harbor the mutation, while 3 developed PGLs so far. Conclusion: Two cases of SDHB-related malignant PGL diagnosed at early ages were presented: one apparently sporadic (P1) and one familial (P2). Despite aggressive disease, with multiple disseminated bone metastases, a long-term survival has been achieved so far, with stable disease after high cumulative I131-MIBG doses and absence of significant adverse effects. Considering the rarity of the disease especially in children, these cases significantly add to the literature regarding the long-term course of malignant PGL and treatment options.

SAT-320 A Synonymous Pathogenic Variant (p.l180=) in SDHB Gene Identified in a Young Patient with Abdominal Paraganglioma

Background: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors derived from chromaffin cells. More than 30% of patients with PPGL have a hereditary predisposition. To date, at least 14 tumor susceptibility genes have been described. Mutations in the succinate dehydrogenase complex iron sulfur subunit B gene (SDHB) are more associated with paragangliomas and metastatic disease. Patients who are young, with larger tumors (>6 cm), positive genetic testing (especially SDHB) or paraganglioma have a higher risk of metastasis development. Case Report: A 14-yr-old boy was referred to investigate severe hypertension and paroxysmal headache, pallor and tachycardia. Biochemical testing for catecholamine-secreting PPGLs showed: 24h urine metanephrines 1.9 µg (normal range, 0.05 to 1.2), plasma normetanephrine 13.3 nmol/L (<0.9 nmol/L), plasma metanephrine <0.2 nmol/L (<0.5 nmol/L), 24h urine norepinephrine 987 µg (14 to 80), 24h urine epinephrine 0 (0.5 to 20 µg) and 24h urine dopamine 391 µg (65 to 400). Magnetic resonance imaging (MRI) revealed a 6.5 cm heterogenous and high-vascular mass in right adrenal gland topography. A strong positive uptake was evidenced in the metaiodobenzylguanidine (MIBG) scintigraphy. A selective α1-receptor blocker (prazosin) was started to control blood pressure and adrenergic symptoms. The patient underwent laparoscopic surgery and anatomopathological analysis revealed a 5.5 cm paraganglioma (PASS criteria= 3). The normal right adrenal gland was identified in the histopathology and there was no continuity between normal adrenal gland and neoplasia. After one year of follow-up, the patient is in complete remission. Genetic analysis by Sanger automated sequencing revealed no pathogenic variants in VHL and SDHD genes. We identified a heterozygous germline synonymous variant c.540 G>A (p.L180=), involving a G>A transition in the last nucleotide of SDHB exon 5. This synonymous variant results in alternative splicing of the SDHB primary transcript. This germline variant was previously reported in a 12-yr-old patient with isolated jugulotympanic paraganglioma, but it has not been associated with catecolamine-secreting paraganglioma. Neck MRI revealed no abnormalities in our patient. Conclusion: We report here a synonymous pathogenic variant in SDHB exon 5 associated with an abdominal functioning paraganglioma. Annual screening for multifocal disease (abdominal and neck PPGLs) and malignancy should be performed.

Combination of Suspension Array and Mycelial Growth Assay for Detecting Multiple-Fungicide Resistance in Botrytis cinerea in Hebei Province in China.

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of Botrytis cinerea in the field, we used the suspension array, sequencing, and mycelial growth assay in our research. Discriminating-dose bioassays for detecting carbendazim, diethofencarb, boscalid, and iprodione resistance (CarR, DieR, BosR, and IprR, respectively) were used to analyze 257 isolates collected from Hebei Province in China during 2016 and 2017. High resistance frequencies to carbendazim (100%), diethofencarb (92.08%), and iprodione (86.59%) were detected. BosR isolates accounted for 11.67% of the total. In addition, 103 isolates were randomly selected for phenotype and genotype detection. The high-throughput suspension array was utilized to detect eight genotypes simultaneously, including BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (FenR), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the BenA gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All BosR isolates carried the H272R mutation in the SdhB gene. The I365S and Q369P+N373S (66.99%) mutations in the BcOS1 gene were more frequently observed. No mutation was detected in the erg27 gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and CarRDieRBosSIprRFenS was the major type. CarR isolates that carried E198V/K/A were all highly resistant (HR) and only one F200Y mutant was moderately resistant (MR) to carbendazim. Isolates that possessed E198V/K were MR or HR to diethofencarb. BosR isolates that possessed H272R mutation were lowly resistant (LR). IprR isolates were all LR or MR. The distribution of half maximal effective concentrations of CarR isolates with E198V/K mutations and IprR isolates with Q369P+N373S mutations significantly increased from 2016 to 2017. Combined with our observations, a combination method of the high-throughput suspension array and the mycelial growth assay was suggested to accurately monitor multiple resistance of B. cinerea in the field.

Long-Term Efficacy and Safety of Molidustat for Anemia in Chronic Kidney Disease: DIALOGUE Extension Studies

Background: Molidustat, a novel hypoxia-inducible factor-prolyl hydroxylase inhibitor, is being investigated for the treatment of anemia associated with chronic kidney disease (CKD). The efficacy and safety of molidustat were recently evaluated in three 16-week phase 2b studies. Here, we report the results of two long-term extension studies of molidustat. Methods: Both studies were parallel-group, open-label, multicenter studies of ≤36 months’ duration, in patients with anemia due to CKD, and included an erythropoiesis-stimulating agent as active control. One study enrolled patients not receiving dialysis (n = 164), and the other enrolled patients receiving hemodialysis (n = 88). The primary efficacy variable for both studies was change in blood hemoglobin (Hb) level from baseline to each post-baseline visit, and safety outcomes included adverse events (AEs). Results: In patients not on dialysis, the mean ± SD Hb concentrations at baseline were 11.28 ± 0.55 g/dL for molidustat and 11.08 ± 0.51 g/dL for darbepoetin. The mean ± SD blood Hb concentrations throughout the study (defined as mean of each patient’s overall study Hb levels) were 11.10 ± 0.508 and 10.98 ± 0.571 g/dL in patients treated with molidustat and darbepoetin, respectively. Similar proportions of patients reported at least one AE in the molidustat (85.6%) and darbepoetin (85.7%) groups. In patients on dialysis, mean ± SD Hb levels at baseline were 10.40 ± 0.70 and 10.52 ± 0.53 g/dL in the molidustat and epoetin groups, respectively. The mean ± SD blood Hb concentrations during the study were 10.37 ± 0.56 g/dL in the molidustat group and 10.52 ± 0.47 g/dL in the epoetin group. Proportions of patients who reported at least one AE were 91.2% in the molidustat group and 93.3% in the epoetin group. Conclusions: Molidustat was well tolerated for up to 36 months and appears to be an effective alternative to darbepoetin and epoetin in the long-term management of anemia associated with CKD.

The mutational burden of targeted genes significantly correlated with overall survival after targeted therapy in metastatic renal cell carcinoma.

643 Background: This study aimed to find the correlation between tumor mutation burden and systemic first line therapeutic response in metastatic tissue samples from patients with metastatic renal cell carcinoma (mRCC). Methods: Between 2005 and 2017, 168 triplet-tissue block samples from 56 mRCC patients were selected for targeted gene sequencing (TGS) using the 88 targeted genes from the National Cancer Center, Korea (NCC) kidney cancer panel. The patients’ medical records, including therapeutic responsive profiles with overall survival (OS) to first-line targeted therapy, were evaluated with the mutational burden of triplet tissue samples using 88 TGS. A few significant target genes associated with therapeutic response towards targeted therapy were identified after comparing the mutational burden of positive for all three blocks and one or two positive blocks (p-value &lt; 0.05). Results: The median PFS for the first-line targeted therapy and OS were 8.7 and 42 months, respectively. MSKCC and Heng risk criteria showed 28.9/65.8/5.3% and 26,3/57.9/15.8% for favorable, intermediate, and poor risk groups, respectively. Also, 55.3% and 52.6% patients received metastatectomy and nephrectomy, respectively. Eighteen (32.1%) patients had all triplet blocks passed for quality check, whereas 21 (37.5%) and 17 (30.4%) patients had two or one passed tissue blocks, respectively. Among the 18 patients with triplet-block, TP53, URB4, PTK2, and SGO2 genes had significant discrimination power for OS on comparing their mutational burden in the three blocks positive group (N = 7) and two or fewer blocks positive groups (N = 11) (p &lt; 0.05). Among the 39 patients with either doublet or triplet blocks passed for quality check, TP53, URB1, PTK2, SGO2, BRAF, NEDD4, PDXDC1, CDH1, FGFR2, RET, RUNX1, and SDHB genes had significant discrimination power for DFS when comparing their mutational burden in the three blocks positive group (N = 7) and two or fewer blocks positive groups (N = 14) (p &lt; 0.01). Conclusions: The study showed the tumor mutational burden of many vital targeted genes to be significantly correlated with OS from metastatic tissues in mRCC.

Discovery of Novel Thiazole Carboxamides as Antifungal Succinate Dehydrogenase Inhibitors

To contribute molecular diversity for novel fungicide development, a series of novel thiazole carboxamides were rationally designed, synthesized, and characterized with the succinate dehydrogenase (SDH) as target. Bioassay indicated that compound 6g showed the similar excellent SDH inhibition as that of Thifluzamide with IC50 of 0.56 mg/L and 0.55 mg/L, respectively. Some derivatives displayed improved in vitro fungicidal activities against Rhizoctonia cerealis and Sclerotinia sclerotiorum with EC50 of 1.2-16.4 mg/L and 0.5-1.9 mg/L. Surprisingly, 6g showed promising in vitro fungicidal activities against R. cerealis and S. sclerotiorum with EC50 of 6.2 and 0.6 mg/L, respectively, which was superior to Thifluzamide with the EC50 of 22.1 and 4.4 mg/L, respectively. Additionally, compounds 6c and 6g displayed excellent in vivo fungicidal activities against S. sclerotiorum on Brassica napus L. leaves with protective activity of 75.4% and 67.3% at 2.0 mg/L, respectively, while Thifluzamide without activity at 5.0 mg/L. Transcriptomic analysis of S. sclerotiorum treated with 6g by RNA sequencing indicated the down-regulation of succinate dehydrogenase gene SDHA and SDHB, and the inhibition of the TCA-cycle.

Maternal and Infant Supplementation with Small-Quantity Lipid-Based Nutrient Supplements Increases Infants’ Iron Status at 18 Months of Age in a Semiurban Setting in Ghana: A Secondary Outcome Analysis of the iLiNS-DYAD Randomized Controlled Trial

ABSTRACTBackgroundInterventions are needed to address iron deficiency in low-income settings.ObjectiveThis secondary outcome analysis aimed to compare the hemoglobin (Hb) and iron status [zinc protoporphyrin (ZPP)] of children born to women enrolled in the iLiNS-DYAD trial in Ghana.MethodsWomen ≤20 wk pregnant (n = 1320) were assigned to receive 60 mg Fe/d and 400 µg folic acid/d until delivery and placebo thereafter, and no supplementation for infants (IFA group); or multiple micronutrients containing 20 mg Fe/d until 6 mo postpartum and no supplementation for infants (MMN); or small-quantity lipid-based nutrient supplements (SQ-LNSs) containing 20 mg Fe/d until 6 mo postpartum, and SQ-LNSs for infants from 6 to 18 mo of age (LNS). We compared infants’ Hb (g/L) and ZPP (µmol/mol heme) at 6 and 18 mo of age.ResultsAt 6 mo of age, groups did not differ in mean ± SD Hb (overall: 113 ± 9.9 g/L) or geometric mean (95% CI) ZPP [overall: 62.6 (60.6, 64.7)]. At 18 mo of age, mean ± SD Hb (overall: 112 ± 10.4 g/L) did not differ significantly between groups, whereas geometric mean (95% CI) ZPP was lower (P = 0.031) in the LNS group [53.9 (50.7, 57.3)] than the IFA [60.4 (56.7, 64.3)] but not the MMN [58.8 (55.6, 62.2)] group. Further, the LNS group, compared with the IFA and MMN groups combined, had a lower prevalence of elevated (>70) ZPP (27.5% compared with 35%; P = 0.02) and a marginally lower prevalence of anemia (38.7% compared with 44.9%; P = 0.06). These results generally remained unchanged when controlling for prespecified covariates or correcting for inflammation.ConclusionsIn this setting, providing SQ-LNSs or multiple micronutrients with 20 mg Fe/d, compared with iron (60 mg/d) and folic acid, to pregnant women does not affect their infants’ Hb or iron status at 6 mo of age, but maternal and infant supplementation with SQ-LNSs increases infants’ iron status at 18 mo of age.This trial was registered at clinicaltrials.gov as NCT00970866.

Patterns of Mutation Enrichment in Metastatic Triple-Negative Breast Cancer.

Background:Triple-negative breast cancer (TNBC) is a heterogeneous disease with aggressive biology and complex tumor evolution. Our purpose was to identify enrichment patterns of genomic alterations in metastatic triple-negative breast cancer (mTNBC).Methods:Genomic data were retrieved (mutations and copy number variations) from 550 primary TNBC tumors from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) data sets and 58 mTNBC tumors from “Mutational Profile of Metastatic Breast Cancers” and “The Metastatic Breast Cancer Project.” Statistical analysis of microarray data between primary and metastatic tumors was performed using a chi-square test, and the percentage of mutation enrichment in mTNBC cases was estimated. P-values were adjusted for multiple testing with Benjamini-Hochberg method with a false-discovery rate (FDR) <.05. In addition, we identified dominant hallmarks of cancer in mTNBC.Results:Seven genes with mutations were enriched in mTNBC after correcting for multiple testing. These included TTN, HMCN1, RELN, PKHD1L1, DMD, FRAS1, and RYR3. Only RPS6KB2 amplification was statistically significant in mTNBC; on the contrary, deletion of the genes TET1, RHOA, EPHA5, SET, KCNJ5, ABCG4, NKX3-1, SDHB, IGF2, and BRCA1 were the most frequent. The molecular alterations related to the hallmark of “genetic instability and mutation” were predominant in mTNBC. Interestingly, the hallmark of “activating immune destruction” was the least represented in mTNBC.Conclusion:Despite the study limitations, we identified recurrent patterns of genomic alterations with potential contribution to tumor evolution. Deletions were the aberrations more commonly found in mTNBC. Several molecular alterations are potentially targetable.