ObjectiveMutations of genes encoding the four subunits of succinate dehydrogenase (SDH) have been associated with pheochromocytoma and paraganglioma (PPGLs), gastrointestinal stromal tumors (GISTs) and renal cell carcinomas (RCCs). These tumors have not been characterized in a way that reflects severity of SDH dysfunction. Mass spectrometric analysis now allows measurement of metabolites extracted from formalin fixed paraffin embedded (FFPE) specimens. We assess whether SDH deficiency in various tumor types characterized by loss of SDHB protein expression correlates with SDH dysfunction as assessed by the ratio of succinate:fumarate in FFPE specimens.Patients and methodsSections of FFPE tumor specimens from 18 PPGL, 10 GIST and 11 RCC patients with known SDHx mutation status for SDH deficiency were collected for mass spectrometric analysis of succinate and fumarate.ResultsFFPE samples showed higher succinate:fumarate ratios in SDH-deficient PPGLs compared to SDH-sufficient PPGLs. Similarly, a higher succinate:fumarate ratio was able to distinguish SDH-deficient GISTs and RCCs from their SDH-sufficient counterparts with great selectivity. Interestingly, the cut-off value of the succinate:fumarate ratio was two-folds greater in RCCs than GISTs.ConclusionAnalyzing biochemical imbalances preserved in FFPE specimens with mass spectrometry expands the method and sample type repertoire available for characterisation of multiple neoplasias associated with SDH deficiency.

Background Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. Methods 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. Results Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975–1.078), and 1.019 (range 0.963–1.081), and intercepts of −0.584 (−53.736 to 54.790) and −3.194 (−17.152 to 5.933), respectively. The mean bias between methods was 1.2% (−11.6% to 16.0%) for metanephrine and 0.1% (−18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from −32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. Conclusions Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

Metabolomics refers to the detection and measurement of small molecules (metabolites) within biological systems, and is therefore a powerful tool for identifying dysfunctional cellular physiologies. For pheochromocytomas and paragangliomas (PPGLs), metabolomics has the potential to become a routine addition to histology and genomics for precise diagnostic evaluation. Initial metabolomic studies of ex vivo tumors confirmed, as expected, succinate accumulation in PPGLs associated with pathogenic variants in genes encoding succinate dehydrogenase subunits or their assembly factors (SDHx). Metabolomics has now shown utility in clarifying SDHx variants of uncertain significance, as well as the accurate diagnosis of PPGLs associated with fumarate hydratase (FH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH2) and aspartate transaminase (GOT2). The emergence of metabolomics resembles the advent of genetic testing in this field, which began with single-gene discoveries in research laboratories but is now done by standardized massively parallel sequencing (targeted panel/exome/genome testing) in pathology laboratories governed by strict credentialing and governance requirements. In this setting, metabolomics is poised for rapid translation as it can utilize existing infrastructure, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the measurement of catecholamine metabolites. Metabolomics has also proven tractable to in vivo diagnosis of SDH-deficient PPGLs using magnetic resonance spectroscopy (MRS). The future of metabolomics – embedded as a diagnostic tool – will require adoption by pathologists to shepherd development of standardized assays and sample preparation, reference ranges, gold standards, and credentialing.

This study examined the pharmacokinetics and viral genomic data from a prospective cohort of kidney transplant recipients undergoing valganciclovir prophylaxis for cytomegalovirus (CMV) prevention. We showed for the first time using high-throughput sequencing the detection of ganciclovir resistance mutations in breakthrough CMV infection during subtherapeutic plasma ganciclovir as indicated by the pharmacokinetic parameter daily area under the curve (AUC 24 ).

Context Germline mutations in the succinate dehydrogenase genes (SDHA/B/C/D, SDHAF2 - collectively, SDHx) have been implicated in paraganglioma (PGL), renal cell carcinoma (RCC), gastrointestinal stromal tumor (GIST) and pituitary adenoma (PA). Negative SDHB tumor staining is indicative of SDH-deficient tumors, usually reflecting an underlying germline SDHx mutation. However, approximately 20% of individuals with SDH-deficient tumors lack an identifiable germline SDHx mutation. Methods We performed whole exome sequencing (WES) of germline and tumor DNA followed by Sanger sequencing validation, transcriptome analysis, metabolomic studies and haplotype analysis in two Italian-Australian families with SDH-deficient PGLs and various neoplasms, including RCC, GIST and PA. Results Germline WES revealed a novel SDHC intronic variant, which had been missed during previous routine testing, in four affected siblings of the index family. Transcriptome analysis demonstrated aberrant SDHC splicing, with the retained intronic segment introducing a premature stop codon. WES of available tumors in this family showed chromosome 1 deletion with loss of wild-type SDHC in a PGL and a somatic gain-of-function KIT mutation in a GIST. The SDHC intronic variant identified was subsequently detected in the second family, with haplotype analysis indicating a founder effect. Conclusions This is the deepest intronic variant to be reported amongst the SDHx genes. Intronic variants beyond the limits of standard gene sequencing analysis should be considered in patients with SDH-deficient tumors but negative genetic test results.