Approaches to Gene Mutation Analysis Using Formalin-Fixed Paraffin-Embedded Adrenal Tumor Tissue From Patients With Primary Aldosteronism

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal’s capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.

PATH-16. COMPREHENSIVE GENOMIC PROFILING ACCURATELY DETERMINES 1p19q CODELETION STATUS IN GLIOMAS

BACKGROUND Genomic profiling of gliomas is vital to ensure diagnostic accuracy, inform prognosis, and identify therapeutic options for primary and recurrent tumors. The integration of genomic biomarkers into brain tumor classification has improved the diagnostic accuracy and led to the development of molecularly stratified clinical trials. DESIGN Comprehensive genomic profiling (CGP) was performed on FFPE material from 310 (162 FoundationOne® and 148 FoundationOne® CDx) samples of brain tumors with available 1p19q FISH results, initially diagnosed by submitting institutions based on histology. Via CGP, we analyzed tumors in up to 395 cancer-associated genes (including IDH1/2) and predicted 1p19q codeletion using a custom research-use only algorithm. Progression-free (PFS) and overall survival (OS) were determined for 519 patients based on computationally predicted 1p19q codeletion status. RESULTS For all samples, regardless of their IDH1/2 mutation status, concordance between 1p19q status based on FISH and our algorithm was 97.1%, (301/310), with a positive predictive value (PPV) of 100% (133/133) and sensitivity of 93.7% (133/142). All discordant samples were called as positive for codeletion by FISH and negative by our CGP-based algorithm. Discordant samples were either IDH1/2 wild-type (2) or IDH1/2, ATRX, and TP53 altered (7), consistent with the genomic profile of diffuse astrocytomas. For IDH1/2-mutated samples, concordance was 96.7% (238/246). In the clinical outcomes dataset, median PFS was 35 months for the codeletion group compared to 13 months for the non-codeletion group (hazard ratio (HR): 0.60; 95% CI: 0.40-0.88; p=0.009). Similarly, median OS was 160 and 34 months respectively for codeleted versus intact (HR: 0.46; 95% CI: 0.28-0.76; p=0.002). CONCLUSIONS 1p19q codeletion status derived from CGP is highly concordant with FISH results suggesting that CGP-derived 1p19q codeletion status may be a reliable substitute for traditional FISH testing. Patients with CGP-derived 1p19q codeletion showed increased PFS and OS compared to non-codeleted counterparts.

Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. Results. We first compared the calls between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between variant callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to intra-tumor heterogeneity (ITH). Conclusion. This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. Results. We first compared the calls between an FF and an FFPE from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between variant callers. Combining the variants of the different callers using a simple heuristic increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to intra-tumor heterogeneity (ITH). Conclusion. This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

Comparative analysis of somatic variant calling on matched FF and FFPE WGS from a metastatic prostate sample

Background. Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, do collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artifacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same prostate metastatic tumor. Results. We first compared the calls between FF and FFPE, showing that on average 50% of the calls in FF are recovered in FFPE, with notable differences between variant callers. Remarkably, this overlap was better than the overlap between different variant callers on the same sample. Inspecting the Variant Allele Frequency (VAF), we observed that many of the calls common to FF and FFPE belonged to the same clonal subpopulation but were detected at a lower VAF in FFPE. We also demonstrated that these calls receive higher significance scores and are often identified by more than one variant caller. Based on this observation, we propose a simple heuristic to perform reliable variant calling in FFPE samples. Our heuristic identified 3684 common calls at a F1-score of 0.83. Conclusion. This study illustrates that when using the correct variant calling strategy, the overlap between the FF and FFPE sample in somatic SNVs increases to such an extent that a large fraction of the calls detected in the FFPE sample are contained in the FF sample and the number of variants unique to each sample remains restricted. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

A streamlined mass spectrometry-based proteomics workflow for large scale FFPE tissue analysis

ABSTRACTFormalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Here, we describe an MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from pathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including less than 10,000 cancer cells isolated by laser-capture microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total work up took less than a day. We observed a moderate trend towards lower protein identifications in long-term stored samples (>15 years) but clustering into distinct proteomic subtypes was independent of archival time. Our results underline the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and prove the feasibility of analyzing large tissue cohorts in a robust, timely and streamlined manner.