Advances in cellular pathology techniques will improve diagnostic medicine. However, such improvements have to overcome many challenges including variations in pre-analytical sample processing, bioinformatics data analysis and clinical interpretation of data. In order to resolve such challenges, bioinformatics needs to become more tightly coupled to the experimental methodology development

Gandy, M

Hamoudi, R

Kapp, J

Umur, S

UCL Discovery

IntroductionMolecular diagnostics within cellular pathology have been performed since the late 1990s and have developed to include a range of techniques including short tandem repeat (STR) identity analysis, classiﬁ cation of tumours and clonality determinations in hematopathology. More recently, with the introduction of qPCR and more recently of next generation sequencing (NGS) as shown in Figure 1, precision medicine test-ing for targeted therapies has rapidly gained access to daily practice and become a chal-lenge for molecular biologists and patholo-gists to provide the most accurate and relevant information. As part of this testing process we discuss two major challenges which have developed, these are:• Firstly, pre-analytical processing of formalin-ﬁ xed paraﬃ  n-embedded (FFPE) tissue, has shown to be a criti-cal determinant in the accuracy of downstream molecular testing in spe-cialities such as mutational screening for targeted therapies.• Secondly, bioinformatics has become a bottleneck in data processing and inter-pretation, with the processing, analysis and reporting of the data shown vari-ability between diﬀ erent laboratories.Th is article looks to raise the awareness of these issues and presents possible areas for consideration to aid in their resolution.Variation in pre-analytical sample processing of FFPE samples may lead to discrepancies in muta-tional testing of actionable genesWithin cellular pathology, the majority of molecular diagnostic clinical sample testing is now carried out on FFPE samples. Gen-erally the tissue is screened using hematox-ylin and eosin stained sections to estimate the tumour content before the preparation process of material for subsequent molecu-lar testing, as shown in Figure 2.Recent studies have shown that variations in pre-analytical processing of samples lead to discrepancies in downstream molecu-lar diagnostic testing [1–3]. Th e variations using singleplex mutational screening were largely due to the DNA extraction system used [2, 3], quantitation using spectropho-tometry and training of laboratory staﬀ  as one study showed that pre-analytical vari-ation was signiﬁ cant even among experi-enced laboratories [3]. In addition both DNA quantitation and integrity measure-ments play important roles in the accuracy of downstream multiplex testing using NGS.In order to resolve some of those issues it is important to include control series of diag-nostic samples, prepared according to the diagnostic operating procedures of the labo-ratory with a variety of known mutations comprising missense mutations, simple and complex deletions and insertions. Assay con-trol using known representative DNA sam-ples from the FFPE tissue is also essential to ensure that the process of DNA extraction, quantitation and integrity measurements are performed correctly and consistently. Th is is important as DNA quality has a major eﬀ ect on NGS performance, i.e. poor quality DNA causes a higher error rate [3].Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathologyAdvances in cellular pathology techniques will improve diagnostic medicine. However, such improvements have to overcome many challenges including variations in pre-analytical sample processing, bioinformatics data analysis and clinical interpretation of data. In order to resolve such challenges, bioinformatics needs to become more tightly coupled to the experimental methodology development.by Dr Rifat Hamoudi, Dr Joshua Kapp, Sevgi Umur and Michael Gandy – September 2015 Pathology focus16Figure 1. Historical development of sequencing technology.0012_025_CLI_Sep.indd   16 27/08/15   10:51In addition, diﬀ erences in quantitation meas-urements need to be accounted for, since the diﬀ erent instruments used have diﬀ er-ent ways of measuring the concentration of DNA. For example, variations can be seen between systems such as Nanodrop spectro-photometry and Qubit ﬂ uorometry. Meas-urement of DNA integrity is also important and most labs use assays such as BIOMED [4, 5] or qPCR as the ‘gold standard’ measure.Also European external quality assurance (EQA) programmes for mutation detection of solid tumours such as European Society for Pathology (ESP, www.esp-pathology.org), European Molecular Genetics Qual-ity Network (EMQN, www.emqn.org), and United Kingdom National External Qual-ity Assessment Scheme UK NEQAS for Molecular Pathology (www.ukneqas.org.uk and www.ukneqas-molgen.org.uk) may consider including pre-analytical (e.g. pre-PCR) component in their assessment for mutation detection from FFPE samples.Discrepancies in variant-calling pipelines and high-throughput sequencing clinical interpretationMost diseases such as cancer and inherited dis-eases are driven by genomic alterations. Recent advances in high-throughput sequencing technologies have enabled the identiﬁ cation of somatic mutations at very high resolution. However, accurate somatic mutation-calling using high-throughput sequence data remains one of the major challenges in genomics. For somatic mutation-calling, one looks for a site in which a variant allele exists in the tumour sample but not in the normal sample. Even with the sequence data from a normal sample, variant-calling in high-throughput sequencing data is challenging due to the multiple poten-tial sources of errors. For example, artefacts occurring during PCR ampliﬁ cation or tar-geted capture (e.g. exome-capture), machine sequencing errors, and incorrect local align-ments of reads are all well documented sources of error [6–8]. Tumour heterogeneity and normal contamination contribute additional challenges for the tumour samples [9].Various studies have shown low concord-ance between diﬀ erent variant callers and bioinformatics analysis pipelines. Wang et al. [10] compared six variant callers on whole exome sequencing melanoma sample and matched blood of 18 lung tumour–nor-mal pairs and seven lung cancer cell lines carried out on the Illumina HiSeq 2000. Th e results showed discordance between the six variant callers, and the top two performing callers could only detect 86% and 71% of validated mutations respectively. O’Rawe et al. [11] compared the analysis of ﬁ ve diﬀ er-ent Illumina alignment and variant-calling pipelines on 15 exome sequencing data carried out using Illumina HiSeq 2000 and Agilent SureSelect version 2 capture kit at 120X mean coverage. Results showed vari-ant-calling concordance of 57.4% between the ﬁ ve diﬀ erent Illumina pipelines across all 15 exomes with the authors urging more caution when analysing individual genomes in genomic medicine. In addition, com-parison of the two most prominent cancer genome sequencing databases; catalogue of somatic mutations in cancer (COSMIC) [12] and Cancer Cell Line Encyclopaedia (CCLE) [13] revealed marked discrepan-cies in the detection of missense mutations in identical cell lines (57.4% conformity), where the main reason for such discrepancy is inadequate sequencing of GC-rich areas of the exome [14].In addition to the above, various studies have shown discrepancies in the interpreta-tion of genomic data between the clinician and diagnostic laboratory. Shashi et al. [15] tried to follow up the results of 93 patients who underwent exome sequencing. Th ey investigated how the clinical interpretation of the lab results changed the diagnosis and its conformity with it. Overall, the results showed that in 25% of patients (24/93), exome sequencing showed a positive result and in 80% (19/24) of cases, the clinicians agreed with the molecular diagnosis of the lab. However, in 20% of patients reported to be positive by the diagnostic lab, the cli-nicians thought that the suggested molecu-lar diagnosis was not correct. In addition, 5% of patients that were considered nega-tive by the exome lab or had a lower con-ﬁ dence diagnosis, were eventually found to be positive when the exome data was reviewed by clinicians. In summary the results showed 20% false positives and 5% false negatives when comparing the inter-pretation of genomic data between diﬀ er-ent healthcare staﬀ .However, it is worth noting that all the above studies used samples with high molecular weight DNA from cell lines, fresh frozen tis-sue or blood and carrying out the same studies above using FFPE samples has the potential to lead to further discrepancies due to the degraded DNA inherent to those samples increases the variation at the pre-analytical steps resulting in downstream discrepancies in mutational proﬁ l-ing. Th is crates it a big challenge in the devel-opment of bioinformatics pipelines required to produce consistent clinically reliable data.One way to resolve some of the bioinfor-matics related issues is to exchange the raw datasets between laboratories that prefer-entially use diﬀ erent soft ware as part of the soft ware validation process to establish the ability of the various laboratories to detect identical gene mutations. In addition, new soft ware updates need to be validated by analysis of prior NGS datasets covering simple and complex mutations. Finally, raw NGS datasets need to be included in EQA programmes as in silico assessment.ConclusionAlthough the above discussion very brieﬂ y surveys the current landscape in cellular pathology, the future of molecular diag-nostics will undoubtedly develop to include integrated RNA expression analysis, DNA ampliﬁ cation and epigenetics. Each meth-odology will have its own idiosyncrasies and will require the development of new clinically validated bioinformatics pipeline. Additionally, the need for a novel bioinfor-matics system to support integrative analy-sis will become essential. Although previ-ously attempted [16], new systems need to be developed to support integrative high-throughput sequencing analysis.However, before novel bioinformatics soft ware solutions can be devised for big data, concerns about bioinformatics soft ware development need to be addressed. A potential starting point to  – September 201517Figure 2. Formalin-ﬁ xed parafﬁ n-embedded (FFPE) tissue preparation for molecular pathology testing.0012_025_CLI_Sep.indd   17 27/08/15   10:51address this is via supporting new bioinformatics courses that use soft ware engineering, computer programming and mathematical modelling of biological complexity at their core, supporting the education of future bioinformaticians in the art of bioinformatics soft ware development. Th is will help support a change in the current para-digm where much of the current bespoke bio-informatics soft ware today has been developed by local institutions in relative isolation, oft en in conjunction within the framework of a specialist area experimental research program [17].Th e future landscape highly likely see the validation of wet chemistries (laboratory and clinical based) and dry (computational based) experiments carried out in more tightly coupled format than is currently performed, supporting clinical product development in the commer-cial market. Also, the future will see more focus on the development of more eﬃ  cient adaptive algorithms that address the clinical questions, leading to faster analysis and improving the clarity in the interpretation of the data.In conclusion, within cellular pathology the incremental development of pre-analytical processing from FFPE samples coupled with more eﬃ  cient adaptive bioinformatics algo-rithms implementation are key areas of focus and crucial to the further advancement of next generation molecular pathology.References1.  Carrick DM, Mehaﬀ ey MG, Sachs MC, Altekruse S, et al. Robustness of Next Generation Sequencing on older formalin-ﬁ xed paraﬃ  n-embedded tissue. PLoS One 2015; 10: e0127353.2.  Heydt C, Fassunke J, Kunstlinger H, Ihle MA, et al. Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequenc-ing in routine diagnostics. PLoS One 2014; 9: e104566.3.  Kapp JR, Diss T, Spicer J, Gandy M, et al. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial. J Clin Pathol. 2015; 68: 111–118.4.  Johnson NA, Hamoudi RA, Ichimura K, Liu L, et al. Appli-cation of array CGH on archival formalin-ﬁ xed paraﬃ  n-embedded tissues including small numbers of microdis-sected cells. Lab Invest. 2006; 86: 968–978.5.  van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobu-lin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Con-certed Action BMH4-CT98–3936. Leukemia 2003; 17: 2257–2317.6.  Meacham F, Boﬀ elli D, Dhahbi J, Martin DI, et al. Iden-tiﬁ cation and correction of systematic error in high-throughput sequence data. BMC Bioinformatics 2011; 12: 451.7.  Nakamura K, Oshima T, Morimoto T, Ikeda S, et al. Sequence-speciﬁ c error proﬁ le of Illumina sequencers. Nucleic Acids Res. 2011; 39: e90.8.  Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011; 12: 443–451.9.  Gerlinger M, Rowan AJ, Horswell S, Larkin J, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366: 883–892.10.  Wang Q, Jia P, Li F, Chen H, et al. Detecting somatic point mutations in cancer genome sequencing data: a com-parison of mutation callers. Genome Med. 2013; 5: 91.11.  O’Rawe J, Jiang T, Sun G, Wu Y, et al. Low concordance of multiple variant-calling pipelines: practical implica-tions for exome and genome sequencing. Genome Med. 2013; 5: 28.12.  Forbes SA, Beare D, Gunasekaran P, Leung K, et al. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015; 43: D805-D811.13.  Barretina J, Caponigro G, Stransky N, Venkatesan K, et al. Th e Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483: 603–607.14.  Hudson AM, Yates T, Li Y, Trotter EW, et al. Discrepan-cies in cancer genomic sequencing highlight opportuni-ties for driver mutation discovery. Cancer Res. 2014; 74: 6390–6396.15.  Shashi V, McConkie-Rosell A, Schoch K, Kasturi V, et al. Practical considerations in the clinical application of whole-exome sequencing. Clin Genet. 2015; doi: 10.1111/cge.12569.16.  Watkins AJ, Hamoudi RA, Zeng N, Yan Q, et al. An inte-grated genomic and expression analysis of 7q deletion in splenic marginal zone lymphoma. PLoS One 2012; 7: e44997.17.  Prins P, de Ligt J, Tarasov A, Jansen RC, et al. Toward eﬀ ective soft ware solutions for big biology. Nat Biotech-nol. 2015; 33: 686–687.The authorsRifat Hamoudi*1 PhD, Joshua Kapp1 MBBS, Sevgi Umur2 BSc and Michael Gandy3 MSc1Division of Surgery and Interventional Sci-ence, University College London, London, UK2Genonymous Sciences, Küçükbak-kalköy, Defne Sokak, Flora Residence Istanbul,Turkey3Health Services Laboratories, 60 Whitfi eld Street, London, UK*Corresponding authorE-mail: r.hamoudi@ucl.ac.uk – September 2015 Pathology focus18POCT and preanalytics to be the themes of Labquality Days 2016Th e Labquality Days Congress will be held at the Messukeskus Expo and Convention Centre in Helsinki on 11th-12th of February 2016. Labquality Days is one of the largest annual congresses in Scandinavia focused on quality and laboratory medicine. Th e con-gress inspires clinical chemistry, laboratory medicine professionals, researchers, healthcare experts, users of point-of-care devices, medical staﬀ  working with quality issues, managers and higher level personnel administra-tion of social- and or healthcare sectors. Th e 2016 congress themes are now announced: Point-of-Care Testing (POCT) and pre-analytics. POCT has already a major role in healthcare workﬂ ow. Test sensitivity or speciﬁ city, price, speed and patient convenience are some heavily discussed topics in scientiﬁ c meetings. In preanalytics, various disciplines such as microbiology, clinical chemistry and hematology have their own characteristic variables. Individual analyses have some unique factors that should also be taken into account in order to obtain reliable results. Labquality Days will bring together leading international speakers and opinion leaders. Th e programme consists of scientiﬁ c lectures and panel discussions. During the congress participants have the opportunity to meet colleagues, share ideas and experience the vast clinical laboratory exhibition.www.labqualitydays.comEVENT PREVIEW0012_025_CLI_Sep.indd   18 27/08/15   10:51

Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathology

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Variations in pre-analytical FFPE sample processing and bioinformatics: challenges for next generation molecular diagnostic testing in clinical pathology

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