news updates on | june 2018 | volume 42 ... · showing increased cancer risk in the rela-tives of...

Also in this issue :

Biomarkers in age-relatedmacular degeneration Pg. 13

NGS approach to identify leukemia-associated mtDNA mutations Pg. 20

Hematology analyser characterizes cells in near-native state

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Automated hemostasis analyser

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The genes that influence breast cancer risk Pg.6

News updates on www.clinlabint.com | June 2018 | Volume 42

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In May it was reported that, owing to an IT error, from 2009 to 2018, approximately 450,000 women aged between 68 and 71 were not recalled for their final mammo-gram appointments in England. Jeremy Hunt, the health secretary has been quoted as saying that between 135 and 270 women “may have had their lives shortened as a result”. The panic-quelling response came very quickly. The Guard-ian newspaper reported that Sir Richard Peto, a professor of medi-cal statistics at Oxford University, had written that there is still sub-stantial uncertainty about the exact ages that mammographic screen-ing should start and end. Addition-ally, a group of academics and GPs wrote a letter to The Times newspa-per saying that the women should not be concerned unless they notice a lump or other symptoms and that the breast cancer screen-ing programme mostly causes more unintended harm than good; many women and doctors avoid breast screening as it has no impact on all-cause death; and that the most dangerous and advanced can-cers are not prevented by screening programmes. Breast cancer chari-ties retorted that mammographic screening remains the best tool available for detecting breast can-cer at an early and therefore more easily treatable stage and we must not forget that the programme does save lives. The UK’s NHS breast screening programme began in 1988 and national coverage was reached in the mid-1990s. How-ever, over twenty years on, there seems to be an increasing body of data to suggest that the ‘accidental’ harm resulting from mammogra-phy because of over-detection and over-treatment of clinically unim-portant lumps has been underes-timated. The often-quoted figure is that for every woman whose life is extended, three receive unnec-essary surgery, chemotherapy or radiotherapy. Hence our ‘best tool available’ seems to be a rather blunt tool. Biomarkers, surely, could

provide the refinement needed to stratify patients according to therapy response. This will enable the delivery of individually tai-lored treatment plans and so will, crucially, prevent the unnecessary administration of chemotherapy and radiotherapy. Work on this

is, of course, underway. The EU-funded RESPONSIFY study in Germany has already led to two parameters being included into German breast cancer guidelines for the treatment of HER2-posi-tive breast cancer. Additionally, a gene-expression panel that predicts

whether chemotherapy will be beneficial for preventing recur-rence is already being used, with some success for the low and high scores. Further work is needed, but perhaps the day is in sight where women will no longer undergo unnecessary chemotherapy.

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– June 20183EDITOR’S LETTER

ContentsFRONT COVER

FEATURES

[6 - 12] BREAST CANCER[6 - 8] BRCA and beyond: the genes that influence breast cancer risk

[10 - 12] Risk factors for development of breast cancer bone metastasis

[13 - 26] MOLECULAR DIAGNOSTICS[13 - 17] Biomarkers in age-related macular degeneration

[20 - 23] New NGS sequencing approach of paired diagnostic and remission samples to detect somatic mitochondrial DNA mutations in leukemia

[24 - 26] Direct detection and differentiation of dermatomycosis pathogens by DNA microarray

[30 - 31] PRODUCT HIGHLIGHT Direct HbA1c testing capabilities on the RX series range of clinical chemistry analysers

REGULARS[3] Editor’s letter

[28] News in brief

[29] Industry news

[32 - 34] Product news

[34] Calendar

Over the last twenty-five years, breast cancer ge-netics has moved from linkage in high-risk families to association in population-based studies. Accord-ingly, the genetic variants that have been identi-fied range from rare high-penetrance mutations to common low-penetrance markers. We summarize current knowledge and consider whether under-standing how these variants influence risk could help to refine risk prediction and develop targeted therapies.Also in this issue :

Biomarkers in age-relatedmacular degeneration Pg. 13

NGS approach to identify leukemia-associated mtDNA mutations Pg. 20

Hematology analyser characterizes cells in near-native state

Pg.32


Pg.34

The genes that influence breast cancer risk Pg.6

News updates on www.clinlabint.com |June 2018 | Volume 42

by Beckman Coulter

by DIAsource ImmunoAssays

by Stago


Pg.33

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Rare high-penetrance mutationsThe earliest evidence for genetic suscep-tibility to cancer came from epidemio-logical studies in the 1940s and 1950s showing increased cancer risk in the rela-tives of cancer patients. It was not until the 1990s that linkage analysis, i.e. the genotyping of genetic markers in large family pedigrees, led to the identification of the first breast cancer susceptibility

gene, BRCA1, at 17q21 [1]. Identification of the second breast cancer susceptibility gene, BRCA2, at 13q12-13 followed rela-tively quickly [2]. Mutations in BRCA1 and BRCA2 are present at a frequency of approximately 1 in 800 for BRCA1 and 1 in 500 for BRCA2, they confer high relative risks of breast cancer in carriers (more than tenfold) and are associated with early onset disease [3, 4].

Moderate-risk variantsThe next milestone in breast cancer genet-ics came in 2002 with the discovery of frameshift alteration in the checkpoint kinase 2 gene, CHEK2*1100delC. This variant was discovered using a combina-tion of linkage and mutation screening in a large multiple-case breast cancer family from the Netherlands, followed by analysis of the CHEK2*110delC vari-ant in high-risk breast cancer families, ‘unselected’ breast cancer cases and con-trols [5]. The CHEK2*1100delC variant occurs on a single haplotype indicating that all CHEK2*1100delC-carrying chro-mosomes arise from a single founder; this variant is confined to Northern European populations with a prevalence in controls that varies significantly between North-ern European populations. Compared to truncating mutations in BRCA1 and BRCA2, the relative risk associated with CHEK2*1100delC is moderate – approxi-mately twofold.

Subsequent to the discovery of CHEK2*1100delC, additional moderate-risk variants were identified in candi-date genes including ataxia telangiectasia mutated (ATM), partner and localiser of BRCA2 (PALB2) and BRCA1 inter-acting protein C-terminal helicase 1 (BRIP1). These variants were discovered by sequencing of exons and exon/intron boundaries of DNA damage repair genes in breast cancer cases from high- and moderate-risk families. Variants in these genes occur in the population at com-bined frequencies (per gene) of around 1% and are predominantly protein-trun-cating mutations.

Common low-penetrance variantsIt was not until 2007 that the first genome-wide association study (GWAS) of breast cancer successfully identified five com-mon low-penetrance variants; minor allele frequencies of these variants ranged from 25 to 40% and they were associ-ated with relative risks of 1.07 to 1.26 [6]. Detecting relative risks of this magnitude required three stages of genotyping and a total of 26 258 cases and 26 894 controls. This study was an order of magnitude larger than any previous study mark-ing the beginning of the era of GWAS as

BRCA and beyond: the genes that influence breast cancer risk

from linkage in high-risk families to association in population-

identified range from rare high-penetrance mutations to common low-penetrance markers. We summarize current knowledge and consider whether understanding how these that variants influence risk could help to refine risk prediction and develop targeted therapies.

by Dr Olivia Fletcher and Dr Syed Haider

– June 2018 Breast cancer6

Figure 1. The loge of the breast cancer odds ratio (y-axis) is plotted against the minor allele frequency (MAF, x-axis) for the common low-penetrance variants identified by genome-wide association study (GWAS) and large collaborative studies. The variants identified in the earlier GWAS are indicated in deep blue, the variants identified in the first large pooled analysis (iCOGS) are indicated in turquoise and the variants identified in the most recent pooled analysis (OncoArray) are indicated in green.

well as large consortia. Since 2007 many more breast cancer GWASs have been published, but the major advances in identifying and cataloguing additional low-penetrance variants have come from large collaborative efforts led by the Breast Cancer Association Consortium (http://bcac.ccge.medschl.cam.ac.uk/); in par-ticular two large analyses – the Collabora-tive Oncological Gene-environment study (COGS) and OncoArray [7, 8]. To date, more than 150 low-penetrance variants conferring relative risks of approximately 0.81–1.35 have been identified. Not sur-prisingly, the more common variants with the (relatively) more extreme breast can-cer odds ratios were identified first, by the GWASs (shown in deep blue, Fig. 1); less common variants and variants with less extreme odds ratios were identified most recently, by the largest pooled analysis, OncoArray (shown in green, Fig. 1).

Contribution to the excess familial relative riskBreast cancer, like most common cancers, shows familial aggregation; the risk of breast cancer in the first-degree relative of a breast cancer case is about twice that of the risk in the general population [3]. The proportion of this ‘familial relative risk’ that is explained by one or more variants is the metric used to quantify the relative contributions of the different classes of variants – and to estimate the number of variants that have not yet been identified. Relative proportions of all three types of variants are shown in Figure 2; muta-tions in BRCA1 and BRCA2 account for approximately the same proportion of the familial relative risk as the sum of the common low-penetrance variants.

Differences between coding variants and non-coding variantsOne fundamental difference between the high-penetrance mutations in BRCA1 and BRCA2, the moderate-risk variants in DNA damage repair genes and the low-penetrance variants identi-fied by GWAS is that the vast majority of low-penetrance GWAS variants map to non-coding DNA. Estimating the risk of breast cancer for individual BRCA1 and BRCA2 mutation carriers is not trivial; there is some evidence that breast cancer risks differ according to the position of the mutation within the gene [4] and for BRCA2, there is evidence of effect modi-fication by common low-penetrance variants [9]. For the low-penetrance GWAS variants, however, the problem is rather different; while the relative risks associated with the marker single nucleotide polymorphisms (SNPs) are fairly precisely estimated, the under-lying ‘causal’ variants and the genes that these variants influence remain – largely – unknown. Approaches to the functional characterisation of GWAS risk loci include fine-scale mapping of potentially large genomic regions, the analysis of SNP genotypes in relation to expression of nearby genes (eQTL) and the use of chromatin association methods [chromosome conformation capture (3C) and Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET)] of regulatory regions to determine the identities of target genes. Regulatory elements have been shown to form physical interactions with the genes that they regulate, often over long distances and frequently ‘skipping over’ proximal genes; chromatin association

methods capture these interactions and use them to infer likely target genes. We have recently carried out a high-throughput, high resolution analysis of 63 breast cancer risk loci using Capture Hi-C [10]. We were able to identify 110 putative target genes mapping to 33 risk loci. Although some of these putative target genes were well-known cancer genes others were not; in depth follow-up studies will be required to determine which of these putative target genes truly influence breast cancer risk and the mechanisms by which they do so.

Causal variants and target genes can inform risk prediction and therapyNICE guidelines for the classification, care and management of breast cancer, based on an individual’s family his-tory of breast and other cancers, are used to classify women into three cat-egories: population risk (<17% lifetime risk), moderate risk (17–30% lifetime risk) and high risk (≥30% lifetime risk; https://www.nice.org.uk/guidance/CG164). The options that are available to a woman – increased surveillance, genetic testing, chemoprevention and prophylactic surgery – depend on which category she falls within. A longer-term aim of GWAS is the development of polygenic risk scores (PRS) that can be incorporated into risk prediction algo-rithms to refine risk estimates. A recent analysis based on 77 breast cancer-asso-ciated SNPs, estimated lifetime risks of breast cancer for women in the lowest and highest quintiles of the PRS as 5.3% (population risk) and 17.2% (moder-ate risk), respectively [11]. Inclusion of

– June 20187


larger numbers of SNPs and incorporat-ing causal variants rather than tag SNPs should improve the discriminatory power of the PRS.

In this era of stratified medicine, iden-tifying the genes that underlie GWAS associations and hence – presumably – contribute to defining disease subgroups, also offers the potential for targeted therapies. For instance, metastatic breast cancer patients with germline BRCA1 or BRCA2 mutations who also lack HER2 expression are eligible for Olaparib [a targeted cancer drug that inhibits poly-ADP ribose polymerase (PARP)] as of January 2018. A recent study demon-strated that Olaparib-treated patients have significantly improved progres-sion-free survival (PFS) compared to patients treated with standard-therapy (median PFS of 7 months vs 4.2 months respectively) [12]. Breast cancer patients with germline BRCA1 or BRCA2 muta-tions already have a defect in their DNA repair mechanisms; by blocking PARP proteins, Olaparib acts to exacerbate DNA damage and trigger cell death, spe-cifically in cancer cells (synthetic lethal-ity). Although defects in DNA repair can be a consequence of germline BRCA mutations, some breast cancer patients manifest defects in DNA repair in the absence of germline BRCA mutations; these patients are also regarded as BRCA deficient – a characteristic often termed as ‘BRCAness’ [13]. Scientists are actively

searching for biomarkers of BRCAness in order to assess the suitability of exist-ing PARP inhibitors for patients exhib-iting BRCAness [14]. Additional clini-cal trials on studying efficacy of PARP inhibitors for treating other breast can-cer subgroups are underway.

The associations between GWAS SNPs and disease are very modest, and this is often cited as a disadvantage when it comes to considering the genes that map to these loci as putative drug tar-gets. However, an individual non-cod-ing ‘causal’ SNP will usually explain only a small proportion of variation in expression of the gene(s) that it regu-lates; chemically targeting these genes could have a much more profound effect on disease incidence or outcome. In support of this prediction, a recent investigation by scientists from Glaxo-SmithKline estimated that selecting genetically supported targets (including those identified by GWAS) could dou-ble the success rate of drugs in clinical development. Although this estimate may be less applicable to cancer drugs, where the somatic genome is as impor-tant – or more important – than the germline genome [15,] it leaves open the possibility of new therapies tar-geting the genes that underlie GWAS associations.

AcknowledgementsWe thank Breast Cancer Now for funding this work as part of Programme Funding to the Breast Cancer Now Toby Robins Research Centre.

References1. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994; 266(5182): 66–71.2. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, et al. Identification of the breast cancer susceptibility gene BRCA2. Nature 1995; 378(6559): 789–792.3. Easton DF. How many more breast cancer predisposition genes are there? Breast Cancer Res 1999; 1(1): 14–17.4. Kuchenbaecker KB, Hopper JL, Barnes DR, Phillips KA, Mooij TM, Roos-Blom MJ, Jervis S7, van Leeuwen FE5, Milne RL, et al. Risks of breast, ovarian, and contralateral breast can-cer for BRCA1 and BRCA2 mutation carriers. JAMA 2017; 317(23): 2402–2416.5. Meijers-Heijboer H, van den Ouweland A, Klijn J, Wasielewski M, de Snoo A, Oldenburg R, Hollestelle A, Houben M, Crepin E, et al.

Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations. Nat Genet 2002; 31(1): 55–59.6. Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson D, Ballinger DG, Struewing JP, Morrison J, Field H, et al. Genome-wide asso-ciation study identifies novel breast cancer susceptibility loci. Nature 2007; 447(7148): 1087–1093.7. Michailidou K, Hall P, Gonzalez-Neira A, Ghoussaini M, Dennis J, Milne RL, Schmidt MK, Chang-Claude J, Bojesen SE, et al. Large-scale genotyping identifies 41 new loci associ-ated with breast cancer risk. Nat Genet 2013; 45(4): 353–361e2.8. Michailidou K, Lindstrom S, Dennis J, Bees-ley J, Hui S, Kar S, Lemaçon A, Soucy P, Glubb D, et al. Association analysis identifies 65 new breast cancer risk loci. Nature 2017; 551(7678): 92–94.9. Gaudet MM, Kirchhoff T, Green T, Vijai J, Korn JM, Guiducci C, Segrè AV, McGee K, McGuffog L, et al. Common genetic variants and modification of penetrance of BRCA2-associated breast cancer. PLoS Genet 2010; 6(10): e1001183.10. Baxter JS, Leavy OC, Dryden NH, Maguire S, Johnson N, Fedele V, Simigdala N, Martin LA, Andrews S, et al. Capture Hi-C identifies puta-tive target genes at 33 breast cancer risk loci. Nat Commun 2018; 9(1): 1028.11. Mavaddat N, Pharoah PD, Michailidou K, Tyrer J, Brook MN, Bolla MK, Wang Q, Den-nis J, Dunning AM, et al. Prediction of breast cancer risk based on profiling with common genetic variants. J Natl Cancer Inst 2015; 107(5): pii: djv036.12. Robson M, Im SA, Senkus E, Xu B, Dom-chek SM, Masuda N, Delaloge S, Li W, Tung N, et al. Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N Eng J Med 2017; 377(6): 523–533.13. Lord CJ, Ashworth A. BRCAness revisited. Nat Rev Cancer 2016; 16(2): 110–120.14. Davies H, Glodzik D, Morganella S, Yates LR, Staaf J, Zou X, Ramakrishna M, Martin S, Boyault S, et al. HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on muta-tional signatures. Nat Med 2017; 23(4): 517–525.15. Nelson MR, Tipney H, Painter JL, Shen J, Nicoletti P, Shen Y, et al. The support of human genetic evidence for approved drug indications. Nat Genet 2015; 47(8): 856–860.

The authorsOlivia Fletcher* PhD, Syed Haider PhDBreast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW3 6JB, UK

*Corresponding authorE-mail: [email protected]


Figure 2. The proportion of the familial excess risk that is explained by each category of risk variants is shown as a bar chart.

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IntroductionInvasive breast cancer is diagnosed in over 55 000 women every year within the UK [1]. Despite recent advances in breast cancer treatment around 10 000 women die from breast cancer in the UK annually, almost all as a result of meta-static spread, which can occur years after apparently successful initial treatment. Over 70% of all advanced breast cancer patients develop metastatic spread to the skeleton [2, 3]. Disseminated tumour cells within bone can remain dormant for many years before finally becoming reactivated, leading primarily to bone resorption (osteolytic lesions), but also to unbalanced bone formation in response (osteoblastic lesions). Current treatments to reduce/prevent the skeletal complica-tions in patients with established breast cancer bone metastasis (BCBM) involve the use of antiresorptive agents such as bisphosphonates [such as zoledronic acid (ZA)] [4]. An antiresorptive treat-ment has also been developed which utilizes antibodies directed towards key molecules within BCBM-induced bone destruction, i.e. denosumab [5]. These antiresorptive agents have been highly effective in improving quality of life for patients with BCBM, but do not improve survival once metastasis is established.

Recently, however, large studies have shown that bisphosphonates given as adjuvant treatment in early breast cancer, alongside other standard treatments, lead to a reduction in the numbers of post-menopausal patients developing bone metastasis [6]. Adjuvant treatment also leads to improved overall survival and adjuvant bisphosphonate therapy is now entering standard practice. However, these treatments are not without side effects, including osteonecrosis of the jaw

[7, 8]. Since only a minority of women will develop bone metastasis, biomarkers are required to identify those patients at highest risk, enabling therapy to be tar-geted to those who will benefit, sparing those who will not.

Risk factorsClinicopathological and demo-graphic risk factorsBreast cancer is a heterogeneous disease and pathological staging and grading systems are widely used in routine prac-tice. Although not generally specific for indicating risk of bone metastasis, these systems do categorize patients into sub-groups that determine appropriate treat-ment and risk of progression. The human epidermal growth factor receptor 2 (HER2) and estrogen receptor (ER) have both prognostic and predictive value and are routinely measured. ER is a hormone-regulated nuclear transcription factor that binds estrogen, with consequent expres-sion of genes including the progesterone receptor (PR). Patients with HER2-posi-tive breast cancer have a poorer progno-sis, but targeted treatments are now avail-able. Like ER, HER2 is also a predictive marker, identifying patients who are likely to respond to targeted treatments.

Histological subtype, tumour grade, lymph node involvement and body-mass index all impact on the general risk of metastasis and, therefore, of BCBM. It is well-recognized that bone metastases more commonly develop in ER-positive patients; they can also occur in ER-nega-tive patients. Although these pathological categories are routinely examined, there has been a recent strong research empha-sis upon the discovery of molecular risk factors for development of metastasis, including BCBM.

Molecular risk factors for bone metastasisGenetic risk factorsThere is good evidence that the risk of breast cancer spread to bone can be pre-dicted both on the basis of the intrinsic genetic subtype of the primary tumour as well as the presence of recently identified bone metastasis genes.

Breast cancers can be classified into five intrinsic subtypes – luminal A, luminal B, HER2 enriched, basal-like and normal-like. Luminal-subtype tumours metasta-size predominantly to bone [9, 10]. Basal-like tumours metastasize predominantly to the lymph-nodes, brain and lung, with bone being a relatively infrequent site of metastatic spread [9]. In this way, intrinsic tumour subtypes, which reflect the expres-sion of multiple genes, can influence the probability of breast cancer spread to dif-ferent target tissues.

Genes that predict BCBM have been discov-ered using de novo unbiased genetic screen-ing approaches – including gene copy-num-ber analysis (CNA) – to identify regions of gene amplification specific to BCBM. In one such study, bone-homing variants of breast cancer cells were isolated by repeated intracardiac injection within immunocom-promised mice and isolation of metastatic cells from bone [11]. Comparison of the parental and bone-homing cells identified a genetic region, 16q23, amplified within the bone-homing cells which encoded the gene for the musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factor [11]. Further studies identified the role of MAF as a transcriptional regulator of parathyroid hormone-related protein (PTHrP) – a key regulator molecule within the vicious cycle of bone destruction within BCBM [6]. The MAF-status of primary tumours has the ability to predict the benefit of ZA treatment [12]. Patients with MAF-negative tumours have increased disease-free survival upon ZA treatment compared to control patients; however, the beneficial effects of ZA treat-ment are not observed in patients with MAF-positive tumours [12].

Breast cancer cells which have metasta-sized to bone frequently remain dormant for many years as disseminated tumour

Risk factors for development of breast cancer bone metastasisBreast cancer bone metastasis results in a significant reduction in patient quality of life and upon metastatic spread the disease is considered incurable. Molecules have been identified which predict the risk of developing bone metastases. This review discusses these key molecules and their potential utility within patient treatment decisions.

by Dr Steven L. Wood and Prof. Janet E. Brown


cells (DTCs). Growth signals that are still not completely understood trigger even-tual activation of these DTCs and the formation of macro-metastatic lesions. In a recent study using functional genetic screening a protein kinase [mitogen and stress-activated kinase-1 (MSK1)] has been identified, which in ER-positive breast cancer cells promotes breast cancer cell differentiation and inhibits migration to bone [13]. This suggests that the level of expression of MSK1 within ER-positive breast cancer cells could be used to strat-ify patients in terms of risk of developing BCBM.

Protein-expression risk factors within BCBMSeveral studies have focused on altered pro-tein expression within BCBM. Immunohis-tochemical measurement of the levels of cyclo-oxygenase-2 (COX2), cytokeratin-5/6 (CK5/6), C-X-C chemokine receptor-4 (CXCR4), parathyroid hormone receptor-1 (PTHR1), osteoprotogerin (OPN) and calcium-sensing receptor (CaSR) within primary patient tumours evaluated their potential as potential predictors of the sub-sequent development of BCBM [14]. The absence of cytoplasmic OPN in this study was observed to be an independent risk fac-tor for the development of BCBM, whereas expression of PTHR1 was observed to be associated with BCBM; however, the asso-ciation was not significant within multivar-iate analysis, thus PTHr1 levels are not an independent predictor of BCBM [14].

Quantitative proteomic analysis of paren-tal MDA-MB-231 triple-negative breast

cancer cells and comparison with a bone-homing variant of these cells isolated by repeated intracardiac injection within immunocompromised mice, identified two proteins as predictive of development of BCBM: PDZ-domain containing pro-tein (GIPC1) and macrophage capping-protein (CAPG) [15]. In rigorous adjusted Cox regression analyses, high expression of both CAPG and GIPC1 within primary tumours was associated with a higher risk for development of BCBM within both a training set (n=427) and a subsequent validation set (n=297) of patients selected from the large randomized AZURE trial of adjuvant ZA (AZURE-ISRCTN79831382) [15]. GAPGhigh/GIPC1high status was not associated with development of bone metastasis following ZA treatment suggest-ing that these two markers are also predic-tive of treatment benefit.

Bone morphogenetic protein-7 (BMP7) is a cytokine which can elicit diverse signalling outcomes within breast cancer cells, includ-ing altering the rates of cell migration, inva-sion and apoptosis, as well as its role in bone formation [16]. In a study of the level of expression of BMP7 within breast cancer primary tumours, high expression of BMP7 correlated with a reduced time to develop-ment of BCBM within invasive ductal car-cinomas [17]. In this study BMP7 levels did not correlate with time to BCBM within invasive lobular carcinoma [17].

Components of the bone extracellular matrix are potential markers for BCBM risk and several proteins have been studied

in this regard including bone sialoprotein (BSP), osteopontin and osteocalcin [18]. BSP is a component of the bone min-eralized cell-matrix which can perform numerous functions, including integrin-binding and the regulation of angiogenesis [19]. Serum levels of BSP were observed to be higher in patients with bone-only metastasis of breast cancer compared to patients with both osseous and visceral metastases within both univariate and multivariate analysis, with a circulating BSP concentration of ≥24 ng/ml acting as a significant factor for prediction of BCBM risk [20].

Bone turnover markers to monitor develop-ment of BCBMBone turnover markers are products of active bone resorption and formation. Sev-eral of these markers are products of col-lagen metabolism including procollagen-I N-terminal extension pro-peptide (PINP) and procollagen-I C-terminal extension peptide (PICP) – markers of bone forma-tion, as well as C-terminal type-I collagen telopeptide (CTX) and C-terminal telo-peptide (ICTP) – markers of bone resorp-tion [21]. In a study measuring the lev-els of P1NP, CTX and 1-CTP within 872 patient-serum samples taken at baseline in the AZURE trial of adjuvant ZA, levels of P1NP, CTX and 1-CTP were all found to be prognostic for future BCBM, but none of these markers were prognostic for non-skeletal metastasis overall survival or treat-ment benefit from ZA [22].

In a related study [23], Lipton et al. inves-tigated CTX in 621 postmenopausal early breast cancer patients in a 5-year phase III trial of tamoxifen +/− octreotide. Higher pre-treatment CTX was associated with shorter bone-only recurrence-free survival. However, there was no statistically signifi-cant association with first event in the bone plus concurrent relapse elsewhere or with first recurrence at other distant sites.

In a related study serum levels of total and bone-specific alkaline phosphatase (BSAP), CTX, ICTP, osteocalcin, N-termi-nal telopeptide of collagen (NTX), PINP and tartrate resistant acid phosphatase (TRACP5b; a marker of bone resorp-tion), were measured in postmenopausal women with early stage luminal-type inva-sive ductal carcinoma (IDC) [24]. In this study TRACP5b levels most accurately predicted the development of BCBM, with a 3-marker panel (BSAP, PINP and TRACP5b) serving as an accurate marker panel for BCBM [24].

– June 201811

Figure 1. Breast cancer cells alter the formation and breakdown of bone resulting in the release of bone metabolic markers (including P1CP, NTx and CTx). The ability of breast cancer cells to metastasize to bone is dependent upon both genetic factors within the breast cancer cell (including the MAF transcription factor, the protein kinase MSK1 and the signalling and motility proteins – CAPG, GIPC1, BSP and PTHrP). In addition bone-forming osteoblasts and bone-resorbing osteoclasts harbour key molecules including OPG, RANK, RANKL and TRACP5b which influence the rate of bone turnover. All of these molecules have potential to act as factors for stratification of BCBM risk.

ConclusionThe metastatic spread of breast cancer cells to bone is a multistep process in which cancer cells must enter and survive within the circulation, and then finally leave the circulation and enter (and adapt to) the bone micro-environment. Molecular pro-filing of breast cancer cells at both the genetic and protein level has identified a series of molecules which play pivotal roles in this complex process. As such, differen-tial expression of these molecules within primary patient tumour samples may be used to stratify patients with early breast cancer, in terms of BCBM risk and guiding treatment decisions. To date, the intrin-sic tumour subtype has proven to be the most effective observation predicting risk of BCBM development; however, recent studies have identified new molecular components within bone metastatic breast cancer cells (including key transcription factors and proteins important in cell sig-nalling and cell migration) that may form the basis of future tests.

Once within bone, breast cancer cells trig-ger alterations in the bone micro-environ-ment that favour survival of DTCs. Later when macroscopic metastases form, the altered rates of bone formation and break-down lead to the generation of bone meta-bolic products that can be measured within patients. Altered levels of these bone meta-bolic products predict BCBM development and can also be used to monitor treatment responses. Extracellular matrix compo-nents including BSAP, PINP, TRACP5b, CTX and 1-CTP have proven particularly useful in this regard.

Studies to date have occasionally produced conflicting results. This may reflect the use of widely differing sample sources (rang-ing from animal model systems to patient-derived samples), as well as variations in the patient cohorts used for different clini-cal studies. Despite these limitations, key molecules are becoming evident that can be measured and used to predict the risk of BCBM. Future studies using these candi-date molecules in larger, multicentre clini-cal trials will further refine a testing panel for prediction of BCBM risk.

References1. Cancer Research UK (CRUK). Breast cancer statis-tics (http: //www.cancerresearchuk.org/health-pro-fessional/cancer-statistics/statistics-by-cancer-type/breast-cancer).2. Scheid V, Buzdar AU, Smith TL, Hortobagyi GN. Clinical course of breast cancer patients with osseous metastasis treated with combination chemotherapy.

Cancer 1986; 58(12): 2589–2593.3. Coleman RE. Metastatic bone disease: clinical fea-tures, pathophysiology and treatment strategies. Can-cer Treat Rev 2001; 27(3): 165–176.4. Wilson C, Bell R, Hinsley S, Marshall H, Brown J, Cameron D, Dodwell D, Coleman R. Adjuvant zoledronic acid reduces fractures in breast cancer patients; an AZURE (BIG 01/04) study. Eur J Cancer 2018; 94: 70–78.5. Lipton A, Fizazi K, Stopeck AT, Henry DH, Smith MR, Shore N, Martin M, Vadhan-Raj S, Brown JE, et al. Effect of denosumab versus zoledronic acid in pre-venting skeletal-related events in patients with bone metastases by baseline characteristics. Eur J Cancer 2016; 53: 75–83.6. Guise TA, Kozlow WM, Heras-Herzig A, Padalecki SS, Yin JJ, Chirgwin JM. Molecular mechanisms of breast cancer metastases to bone. Clin Breast Cancer 2005; 5 Suppl(2): S46–53.7. Stopeck AT, Fizazi K, Body JJ, Brown JE, Carducci M, Diel I, Fujiwara Y, Martín M, Paterson A, et al. Safety of long-term denosumab therapy: results from the open label extension phase of two phase 3 studies in patients with metastatic breast and prostate cancer. Support Care Cancer 2016; 24(1): 447–455.8. Rathbone EJ, Brown JE, Marshall HC, Collinson M, Liversedge V, Murden GA, Cameron D, Bell R, Spens-ley S, et al. Osteonecrosis of the jaw and oral health-related quality of life after adjuvant zoledronic acid: an adjuvant zoledronic acid to reduce recurrence trial subprotocol (BIG01/04). J Clin Oncol 2013; 31(21): 2685–2691.9. Huber KE, Carey LA, Wazer DE. Breast cancer molecular subtypes in patients with locally advanced disease: impact on prognosis, patterns of recurrence, and response to therapy. Semin Radiat Oncol 2009; 19(4): 204–210.10. Ignatov A, Eggemann H, Burger E, Ignatov T. Pat-terns of breast cancer relapse in accordance to bio-logical subtype. J Cancer Res Clin Oncol 2018; doi: 10.1007/s00432-018-2644-2.11. Pavlovic M, Arnal-Estape A, Rojo F, Bellmunt A, Tarragona M, Guiu M, Planet E, Garcia-Albéniz X, Morales M, et al. Enhanced MAF oncogene expres-sion and breast cancer bone metastasis. J Natl Cancer Inst 2015; 107(12): djv256.12. Coleman R, Hall A, Albanell J, Hanby A, Bell R, Cameron D, Dodwell D, Marshall H, Jean-Mairet J, et al. Effect of MAF amplification on treatment outcomes with adjuvant zoledronic acid in early breast cancer: a secondary analysis of the interna-tional, open-label, randomised, controlled, phase 3 AZURE (BIG 01/04) trial. Lancet Oncol 2017; 18(11): 1543–1552.13. Gawrzak S, Rinaldi L, Gregorio S, Arenas EJ, Salva-dor F, Urosevic J, Figueras-Puig C, Rojo F, Del Barco Barrantes I, et al. MSK1 regulates luminal cell differ-entiation and metastatic dormancy in ER(+) breast cancer. Nat Cell Biol 2018; 20(2): 211–221.14. Winczura P, Sosinska-Mielcarek K, Duchnowska R, Badzio A, Lakomy J, Majewska H, Pęksa R, Pieczyńska B, Radecka B, et al. Immunohistochemi-cal Predictors of Bone Metastases in Breast Cancer

Patients. Pathol Oncol Res 2015; 21(4): 1229–1236.15. Westbrook JA, Cairns DA, Peng J, Speirs V, Hanby AM, Holen I, et al. CAPG and GIPC1: breast can-cer biomarkers for bone metastasis development and treatment. J Natl Cancer Inst 2016; 108(4): doi: 10.1093/jnci/djv360.16. Alarmo EL, Parssinen J, Ketolainen JM, Savi-nainen K, Karhu R, Kallioniemi A. BMP7 influences proliferation, migration, and invasion of breast can-cer cells. Cancer Lett 2009; 275(1): 35–43.17. Alarmo EL, Korhonen T, Kuukasjarvi T, Huhtala H, Holli K, Kallioniemi A. Bone morphogenetic pro-tein 7 expression associates with bone metastasis in breast carcinomas. Ann Oncol 2008; 19(2): 308–314.18. Bahrami A, Hassanian SM, Khazaei M, Hasanza-deh M, Shahidsales S, Maftouh M, Ferns GA, Avan A. The therapeutic potential of targeting tumor micro-environment in breast cancer: rational strategies and recent progress. J Cell Biochem 2018; 119(1): 111–122.19. Bouleftour W, Granito RN, Vanden-Bossche A, Sabido O, Roche B, Thomas M, Linossier MT, Aubin JE, Lafage-Proust MH, et al. Bone shaft revasculariza-tion after marrow ablation is dramatically accelerated in BSP-/- mice, along with faster hematopoietic recol-onization. J Cell Physiol 2017; 232(9): 2528–2537.20. Bellahcene A, Kroll M, Liebens F, Castronovo V. Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development. J Bone Miner Res 1996; 11(5): 665–670.21. Glendenning P, Chubb SAP, Vasikaran S. Clinical utility of bone turnover markers in the management of common metabolic bone diseases in adults. Clin Chim Acta 2018; 481: 161–170.22. Brown J, Rathbone E, Hinsley S, Gregory W, Gossiel F, Marshall H, et al. Associations between serum bone biomarkers in early breast cancer and development of bone metastasis: results from the AZURE (BIG01/04) trial. J Natl Cancer Inst 2018; doi: 10.1093/jnci/djx280.23. Lipton A, Chapman JA, Demers L, Shepherd LE, Han L, Wilson CF, Pritchard KI, Leitzel KE, Ali SM, Pollak M. Elevated bone turnover predicts for bone metastasis in postmenopausal breast cancer: results of NCIC CTG MA.14. J Clin Oncol 2011; 29(27): 3605–3610.24. Lumachi F, Basso SM, Camozzi V, Tozzoli R, Spaziante R, Ermani M. Bone turnover markers in women with early stage breast cancer who developed bone metastases. A prospective study with multivari-ate logistic regression analysis of accuracy. Clin Chim Acta 2016; 460: 227–230.

The authorsSteven L. Wood MA, PhD; Prof. Janet E. Brown* BMedSci, MB BS, MSc, MD, FRCPAcademic Unit of Clinical Oncology, Depart-ment of Oncology and Metabolism,University of Sheffield, UK



– June 201813Molecular diagnostics

IntroductionAge-related macular degeneration (AMD) is a slow and progressive disease of the macula, i.e. the central part of the retina, and the leading cause of irreversible visual loss in the Western world. Globally, AMD accounts for 8.7% of all blindness and is

predicted to affect 196 million people by 2020; it is more prevalent in populations of European descent than those of Asian and African descent [1]. With the loss of central vision frequently involving both eyes, AMD is a debilitating condition affecting daily tasks such as reading and

driving, and ultimately having severe con-sequences on independence and quality of life. AMD is a late-onset disease with a complex etiology. Major risk factors contributing to susceptibility include age, family history (genetics), light exposure and smoking [2–4].

AMD can be considered a multifactorial dysfunction of the retinal photorecep-tor cells and their support system, which includes the retinal pigment epithelium (RPE), Bruch’s membrane (BrM), and the choroidal vasculature. The fundamental cause of vision loss in AMD is the pro-gressive damage to photoreceptors, which can be triggered by RPE dysfunction and atrophy, impaired transport of oxygen, nutrients and metabolites between vessels and outer retinal cells and leakage from

Biomarkers in age-related macular degenerationAge-related macular degeneration is a late-onset disease of the eye macula that can result in blindness and in a significant deterioration of quality of life. Genetics and oxidative stress from light exposure and smoking are major risk factors. In this brief report, we discuss genetic and plasma epigenetic biomarkers that are examined for their association with the disease.

by Prof. Christos Kroupis, Prof. George Kitsos, Prof. Marilita M. Moschos and Prof. Michael B. Petersen

Figure 1. (a) Normal retina and (b) AMD pathology with drusen, CNV and GA. (Figure from Fritsche LG, Fariss RN, Stambolian D, Abecasis GR, Curcio CA, Swaroop A. Age-related macular degeneration: genetics and biology coming together. Annu Rev Genomics Hum Genet 2014; 15: 151–171. Reproduced with permission).

(a)

(b)

choroidal capillaries that invade the retina through the RPE [5].

Light entering the eye is focused on the retina, where delicately specialized rod and cone photoreceptors allow its trans-duction into chemical signals to visual centers in the brain. Photoreceptors are metabolically active neurons with oxygen requirements that are among the high-est in the human body. In humans, rods and cones exhibit a distinct topography; the macula (6-mm diameter) contains a cone-dominated fovea (0.8-mm diam-eter) that is associated with high-acuity vision [5] (Fig. 1a). Just posterior to the photoreceptors, the RPE consists of polar-ized epithelial cells located at the base of the retina as a single layer of hexagonal cells that are densely packed with pig-ment granules (melanosomes). The RPE is firmly attached to the underlying base-ment membrane (BrM). The RPE pro-vides the nutrients needed to maintain visual function by light-sensitive outer segments of the photoreceptors. RPE melanosomes absorb excess incoming light, which protects the retina from light damage. Other critical roles for the RPE involve phagocytosing shed outer retinal segments and scavenging photoreceptor debris, thus, serving as part of the waste-disposal system for the retina. The RPE is known to produce and to secrete a vari-ety of growth factors to help build and sustain the choroid and photoreceptors [6]. The choroid is an extensive vascular

meshwork of capillaries lining the poste-rior part of the eye that supplies nutrients utilized by the retina and acts as a con-duit for the by-products of photorecep-tor and RPE metabolism [5]. The inner aspect of the choroid, next to the RPE, is the BrM, a laminar extracellular matrix composed mainly of collagen and elas-tin. Accumulating evidence suggests that the molecular, structural and functional properties of the BrM are dependent on age, genetics, environmental factors, reti-nal location and disease state. As a result, some properties of the BrM are unique to each human individual at a given age and, therefore, affect uniquely the progression of AMD [6].

AMD pathologyThere are two AMD forms: dry (in 90% of patients) and wet (in 10%). In the dry form of AMD, apoptosis of the RPE, neu-roretina and choriocapillaris progresses slowly and causes permanent central vision loss. Initially, the BrM exhibits increased deposition of cholesterol and calcium with age. Drusen genesis is a sign of AMD progression (Fig. 1b). Drusen are amorphic extracellular deposits of lipids, proteins, inflammatory molecules in the space between RPE and BrM. The alter-native complement path is activated by lipofuscin constituents (which are mostly by-products of the retinal vision cycle) as a response to the inflammatory process connected with drusen genesis. Unfortu-nately, as we age, mitochondrial function

decreases (and mtDNA mutations accu-mulate) and, therefore, oxidative damage increases. In parallel, antioxidant capac-ity decreases and the efficiency of repair systems and cytoprotective ubiquitin proteolytic system become impaired [4]. Environmental factors associated with increased production of reactive oxygen species (ROS), such as increased light exposure and cigarette smoking, are addi-tive and have been linked with AMD risk. Collectively, these factors create an envi-ronment in which proteins, DNA and lipids become oxidatively damaged. The combination of inadequately neutral-ized oxidized proteins in the drusen and inflammation associated with OSEs (oxi-dative specific epitopes) induce focal loss of RPE cells, degeneration of the over-lying photoreceptors and vision loss as described in Figure 2 [4].

In the advanced dry form of AMD, geo-graphic atrophy (GA) develops from large, confluent drusen proceeds to hyper-pigmentation and then, to cell apoptosis. At present, there is no effective treatment of the dry form. In the wet form, the cause of potential central vision loss is choroidal neovascularization (CNV). An inflamma-tory reaction initiates pathological angio-genesis that penetrates through defects in the BrM and the RPE layers to the subretinal space, where exudation and bleeding destroy photoreceptors. Com-monly used anti-VEGF factors given in repeated intravitreal injections inhibit

– June 2018 Molecular diagnostics14

Figure 2. Summary of the potential pathways leading to AMD. A2E, N-retinylidene-N-retinylethanolamine; CEP, carboxyethylpyrrole; ECM, extracellular matrix; NRF2, nuclear factor erythroid-2 related factor 2; OSEs, oxidation-specific epitopes; UPS, ubiquitin proteolytic system. (Adapted from Chiras D, Kitsos G, Petersen MB, Skalidakis I, Kroupis C. Oxidative stress in dry age-related macular degeneration and exfoliation syndrome. Crit Rev Clin Lab Sci 2015; 52: 12–27 [4]; published with permission.

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neovascularization and can stabilize vision acuity in most wet AMD patients.

Genetic biomarkers in AMDIdentification of associated genetic variants can help uncover disease mechanisms and provide entry points for therapy. Linkage of AMD families to 1q32 and the comple-ment factor H (CFH) gene by many groups in 2005, led to the identification of the first common genome-wide significant risk vari-ant, Y402H (rs1061170, g.43097C>T) with variable frequencies across various popu-lations. This SNP (single nucleotide poly-morphism) results in an impaired alterna-tive complement pathway inactivation. This discovery propagated numerous genetic and genomic studies that have contributed to our understanding of the pathological mechanisms contributing to AMD. Nota-bly, the subsequent association of common and rare alleles at or near several additional complement genes (CFH, C2/CFB, C3, CFI and C9) has led to the ‘inflammation hypotheses’, with cumulative evidence from genetics and histopathological studies [3]. Another major non-complement pathway AMD-associated locus lies on chromosome 10q26 (LOC387715) and many studies have demonstrated a strong association between AMD and the ARMS2 gene that encodes

for a small 107-amino acid protein. ARMS2 A69S SNP (rs10490924, g.5270G>T) is a mutation associated with subsequent mito-chondrial dysfunction, ROS generation and accumulation of somatic mitochondrial DNA mutations. These initial promising findings prompted world-wide efforts and culminated in the AMD Gene consortium 2013 study where 19 common variants were associated with the disease in a large num-ber of patients with the use of SNP microar-rays; still the two aforementioned SNPs pos-sessed the highest odds ratios (OR) for AMD development (between 2.4 and 2.7) with some differences in their effect according to their different allele frequencies in vari-ous populations. It was estimated that these 19 variants can explain ~45% of the genetic heterogeneity in AMD patients above 85 years old; the two main AMD associations with CFH and ARMS2 genes account for a significant 25% of the total cases [5]. There-fore, we and other groups have developed fast, high-throughput robust and accurate genotyping assays for their accurate detec-tion (Fig. 3) [7–9]. Early identification of individuals at risk provides an opportunity to prevent or attenuate the AMD disease. Homozygosity for both CFH and ARMS2 risk alleles increases the progression to advanced AMD stages (GA or CNV) to

48% compared to 5% for those carrying wild-type alleles in both genes [10]. Mod-els incorporating these alleles and/or an expanded variant panel along with smoking and body mass index have been the basis for various commercial tests estimating AMD risk, such as RetnaGene (Nicox), Macula/Vita Risk (ArcticDx), Asper Ophthalmics, etc. Potential nutrigenetic antioxidant interventions have been proposed based on CFH and ARMS2 genotypes [11, 12]. In dry AMD where no therapy exists, anti-complement antibodies are in clinical tri-als right now (eculizumab, lampalizumab) and genetic tests providing information for complement polymorphisms could select appropriate patients that could benefit from such therapy.

The largest and latest 2016 AMD Gene Con-sortium study identified additional loci by using an Illumina human core exome array for >12 million variants in 16,144 advanced AMD patients versus 17,832 controls; 52 independently associated common and rare variants were distributed across 34 loci [13]. Now that technological advances permit – with the advent of next-generation sequenc-ing platforms – it would be extremely useful to validate AMD-specific gene-panels for these patients.


Figure 3. (a) Melting curve analysis after a real-time PCR CFH Y402H genotyping assay in the Light Cycler (Roche) platform: a mutant homozygote CC (red curve), a heterozygote CT (green curve) and a wild-type TT sample (grey curve) are shown. (Adapted from Velissari A, Skalidakis I, Oliveira SC, Koutsandrea C, Kitsos G, Petersen MB, Kroupis C. Novel association of FCGR2A polymorphism with age-related macular degeneration (AMD) and development of a novel CFH real-time genotyping method. Clin Chem Lab Med 2015; 53: 1521–15219 [7].) (b) Agarose electrophoresis result of the ARMS2 A69S PCR-RFLP genotyping method. In the first two wells, the MW marker and the PCR blank are shown. Wells 1,3 show homozygote mutant TT samples, well 2 a wild-type GG sample and wells 4,5 heterozygous G/T samples. (Adapted from Sarli A, Skalidakis I, Velissari A, Koutsandrea C, Stefaniotou M, Petersen MB, Kroupis C, Kitsos G, Moschos MM. Investigation of associations of ARMS2, CD14, and TLR4 gene polymorphisms with wet age-related macular degeneration in a Greek population. Clin Ophthalmol 2017; 11: 1347–1358 [8]).

(a)

(b)

Plasma epigenetic biomarkers in AMDA small, non-coding micro(mi)RNA (18–24 nt) binds to specific mRNAs – depend-ing on its sequence – and results in their degradation by cleavage, translational repression and/or polyA-deadenylation. One miRNA can target many mRNAs but also one mRNA can be targeted by many miRNAs. Emerging evidence aris-ing from tissue studies suggest that beside environmental and genetic factors, epige-netic mechanisms (such as miRNA regu-lation of gene expression) are relevant to AMD and are providing an exciting new avenue for research and therapy. Sera and plasma (which are easily collected non-invasively) contain cell free DNA, RNA and circulating nucleic acids that can serve as potential biomarkers. The miRNAs identified in human plasma are known to be relatively stable, as they have been found to be resistant to RNase deg-radation. A recent study has identified a plasma miRNA expression profile specific for AMD patients [14]. Plasma miRNA expression was first screened for mul-tiple miRNAs and then, those showing differences between patients and healthy controls were further explored with indi-vidual, specific RT-qPCR assays in a larger number of samples. In another study exploring wet and dry AMD differences in plasma, the miRNA expression analysis revealed increased expression of miR661 and miR3121 in dry AMD patients and miR4258, miR889 and let7 in wet AMD patients compared to controls [15].

References1. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, Wong TY. Global prevalence of age-related macular degen-eration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health 2014; 2: e106–e116.2. Kokotas H, Grigoriadou M, Petersen MB. Age-related mac-ular degeneration: genetic and clinical findings. Clin Chem Lab Med 2011; 49: 601–616.3. Tan PL, Bowes RC, Katsanis N. AMD and the alternative complement pathway: genetics and functional implications. Hum Genomics 2016; 10: 23.4. Chiras D, Kitsos G, Petersen MB, Skalidakis I, Kroupis C. Oxidative stress in dry age-related macular degeneration and exfoliation syndrome. Crit Rev Clin Lab Sci 2015; 52: 12–27.5. Fritsche LG, Fariss RN, Stambolian D, Abecasis GR, Curcio CA, Swaroop A. Age-related macular degeneration: genet-ics and biology coming together. Annu Rev Genomics Hum Genet 2014; 15: 151–171.6. Bhutto I, Lutty G. Understanding age-related macular degeneration (AMD): relationships between the photorecep-tor/retinal pigment epithelium/Bruch’s membrane/chorio-capillaris complex. Mol Aspects Med 2012; 33: 295–317.7. Velissari A, Skalidakis I, Oliveira SC, Koutsandrea C, Kitsos

G, Petersen MB, Kroupis C. Novel association of FCGR2A polymorphism with age-related macular degeneration (AMD) and development of a novel CFH real-time genotyp-ing method. Clin Chem Lab Med 2015; 53: 1521–15219.8. Sarli A, Skalidakis I, Velissari A, Koutsandrea C, Stefani-otou M, Petersen MB, Kroupis C, Kitsos G, Moschos MM. Investigation of associations of ARMS2, CD14, and TLR4 gene polymorphisms with wet age-related macular degen-eration in a Greek population. Clin Ophthalmol 2017; 11: 1347–1358.9. Xu Y, Guan N, Xu J, Yang X, Ma K, Zhou H, Zhang F, Snel-lingen T, Jiao Y, et al. Association of CFH, LOC387715, and HTRA1 polymorphisms with exudative age-related macu-lar degeneration in a northern Chinese population. Mol Vis 2008; 14: 1373–1381.10. Seddon JM, Francis PJ, George S, Schultz DW, Rosner B, Klein ML. Association of CFH Y402H and LOC387715 A69S with progression of age-related macular degeneration. JAMA 2007; 297: 1793–1800.11. Awh CC, Hawken S, Zanke BW. Treatment response to antioxidants and zinc based on CFH and ARMS2 genetic risk allele number in the Age-Related Eye Disease Study. Ophthal-mology 2015; 122: 162–169.12. Vavvas DG, Small KW, Awh CC, Zanke BW, Tibshirani RJ, Kustra R. CFH and ARMS2 genetic risk determines progres-sion to neovascular age-related macular degeneration after antioxidant and zinc supplementation. Proc Natl Acad Sci U S A 2018; 115: E696–E704.13. Fritsche LG, Igl W, Bailey JN, Grassmann F, Sengupta S, Bragg-Gresham JL, Burdon KP, Hebbring SJ, Wen C, et al. A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat Genet 2016; 48: 134–143.14. Ertekin S, Yıldırım O, Dinç E, Ayaz L, Fidancı SB, Tamer L. Evaluation of circulating miRNAs in wet age-related macular degeneration. Mol Vis 2014; 20: 1057–1066.15. Szemraj M, Bielecka-Kowalska A, Oszajca K, Krajewska M, Goś R, Jurowski P, Kowalski M, Szemraj J. Serum micro-RNAs as potential biomarkers of AMD. Med Sci Monit 2015; 21: 2734–2742.

The authorsChristos Kroupis*1 MSc, PhD; George Kit-sos2 MD, PhD; Marilita M. Moschos3 MD, PhD; Michael B. Petersen4 MD, PhD

1Department of Clinical Biochemistry and Molecular Diagnostics, Attikon University General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece2Department of Ophthalmology, University General Hospital of Ioannina, Ioannina, Greece31st Department of Ophthalmology, “G. Gennimatas” General Hospital, Medical School, National and Kapodistrian Univer-sity of Athens, Athens, Greece4Department of Clinical Genetics, Aalborg University Hospital, Aalborg, Denmark


– June 201817

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IntroductionThe identification of acquired somatic mutations in leukemic samples is of considerable importance for diagnosis and prognostication. In order to iden-tify somatic mutations it is necessary to have a control tissue from the same individual for comparison. Non-hemat-opoietic tissues, such as mesenchymal stromal cells (MSCs) or hair follicles are preferred, but not always available. When patients with leukemia achieve remission, the remission peripheral blood (PB) may be a suitable and eas-ily available control tissue. This article will provide recommendations for the identification of tumour-associated mtDNA somatic mutations, highlight-ing advantages and disadvantages of the method.

mtDNA characteristicsHuman mitochondrial (mt) DNA is a 16 569 bp double-stranded, circular DNA molecule that encodes 13 poly-peptides of the oxidative phosphoryla-tion system (OXPHOS), 22 transfer RNAs and 2 ribosomal RNAs. Several important differences between the mt genome and the nuclear genome com-plicate the study of mtDNA mutations. Ninety-three percent of the sequence consists of coding DNA, introns are absent, the only non-coding region is at the level of the D-loop contain-ing the promoters of the genes and it is maternally inherited. Each cell has a variable number of mitochondria

(typically several hundred) and each mitochondrion contains a variable number of genomes (typically 2–10). Consequently, mtDNA mutations do not follow the pattern of a diploid genome: rather, a cell may have a single mt genotype (homoplasmy) or multiple mt genotypes (heteroplasmy). Hetero-plasmy may be at any frequency, could vary between cells and many variants will be below the limit of detection of Sanger sequencing, and therefore tech-nically difficult to validate [1]. To date, more than 400 mtDNA mutations have been associated with human diseases, most of them being heteroplasmic. Therefore, an accurate determination of the level of heteroplasmy is impor-tant for disease association studies [2].

“Important differences between the mitochondrial (mt) and nuclear genomes complicate the study of

mtDNA mutations”

mtDNA mutations and cancerMtDNA mutations may potentially contribute to a cell to becoming can-cerous, leading to invasion and metas-tasis [3]. Heteroplasmic somatic mtDNA mutations have been reported in hematological neoplasms, including myelodysplastic syndromes, chronic

lymphocytic leukemia, chronic myeloid leukemia (CML), acute myeloid leuke-mia, and acute lymphoblastic leukemia (ALL) [1]. Many cancer types, including leukemia, have a tendency to be highly glycolytic, increasing the production of the reactive oxygen species (ROS), that lead to genomic instability. The mtDNA genome is susceptible to ROS-induced mutations owing to the high oxida-tive stress in the mitochondrion and limited DNA-repair mechanisms [3]. The identification of acquired somatic mutations in leukemic samples is of considerable importance for diagnosis and prognostication. In a study in acute myeloid leukemia, for example, patients with mutated NADH dehydrogenase subunit 4 (ND4) showed greater overall survival than patients with wild-type ND4 [4].

mtDNA somatic mutations: the problem of control tissueMtDNA acquires somatic mutations at a rate 10-fold higher than nuclear DNA, so mtDNA single nucleotide variants (SNVs) accumulate with age, and may be tissue-specific [5]. This means that there is no absolutely reli-able source of ‘germline’ mtDNA, espe-cially in older individuals [1]. Somatic mutations must be distinguished from non-pathogenic germline variants by comparison with a control tissue sam-ple. Non-hematopoietic tissues, such as buccal cells, hair follicles or MSCs are preferred, but not always available. PB cells from a post-treatment remis-sion sample may be used as alternative. This method is widely used for nuclear mutations, but less commonly for mt mutations [1]. Blood samples are read-ily accessible from leukemia patients who achieve morphological remission after treatment. Therefore, a method for the detection of leukemia-associ-ated mtDNA mutations based on com-parison with a remission sample may be useful.

New NGS sequencing approach of paired diagnostic and remission samples to detect somatic mitochondrial DNA mutations in leukemiaMitochondrial DNA mutations (mtDNA) have been described that are associated with leukemia. To identify somatic mutations it is necessary to have a control tissue from the same individual for comparison. In this review we describe a new next-generation sequencing approach to identify leukemia-associated mtDNA mutations by using remission samples as control.

by Dr Ilaria Stefania Pagani


A new approach to identify mtDNA somatic mutations at diagnosis by using remission samples as control tissuePagani IS and colleagues developed a next-generation sequencing (NGS) approach for the identification of leuke-mia-associated mtDNA mutations using samples from CML patients at diagno-sis and in remission following treat-ment with tyrosine kinase inhibitors (TKIs) [1]. This approach could also be applied to both hematopoietic and non-hematopoietic cancers, such as epithelial tumours, in which a tumour biopsy spec-imen can be compared with the normal mucosa.

Twenty-six chronic phase CML patients enrolled in the Australasian Leukae-mia and Lymphoma Group CML9 trial (TIDEL-II; ID: ACTRN12607000325404) [6] took part in the study [6]. PB samples from leucocytes at diagnosis before com-mencing TKI treatment, and remission after 12 months of therapy were com-pared. Hair follicles (n=4), bone marrow MSCs (n=18), or both (n=4) were used as

non-hematopoietic control samples. The comparison of a diagnostic sample with a non-hematopoietic control tissue is the standard method to identify somatic mutations in leukemia [1]. The concord-ance between this classic method and the diagnosis versus remission approach has been investigated.

NGS assay for the mt genomeThe workflow chart is represented in Figure 1. Briefly the genomic DNA (comprising a mixture of nuclear and mtDNA) was extracted by a phenol/chloroform method from PB leukocytes and non-hematopoietic tissues. The mtDNA was amplified by long-range PCR, generating two or three overlap-ping fragments covering the entire mt genome. The PCR amplicons were then pooled at equimolar concentrations and sequencing libraries were prepared using the Nextera XT kit (Illumina). Indexed libraries were multiplexed and run on an Illumina MiSeq instrument using the 600 cycle MiSeq Reagent kit (v3) generating 300-bp paired-end reads [1].

Somatic mutation calling from high-throughput sequencing datasets and validationThe majority of the variant-calling methods in use are based on low-cover-age human re-sequencing data and dip-loid calls with discrete frequencies of interest (0%, 50% or 100%) [7, 8]; how-ever, these assumptions do not apply to mtDNA. The LoFreq software (loFreq-star version 2.11, genome Institute of Singapore; http://csb5.github.io/lof-req/) was chosen because it was devel-oped for viral and bacterial genomes as well as diploid data, and because of its ability to automate comparison with a matched control tissue for the detection of somatic mutations [8]. The revised Cambridge Reference Sequence (rCRS) for the human mt genome (NC_012920) was used as reference sequence to iden-tify SNVs. Tumour tissue (test) and control were then compared to identify somatic mutations specific only for the tumour tissue. Variants in common between the test and the control sample were considered to represent germline polymorphisms or mutations and were

– June 201821

Figure 1. Schematic representation of the pipeline for the sequencing of the mt genome. MSCs, mesenchymal stromal cells; nDNA, nuclear DNA; NGS, next-generation sequencing; TKIs, tyrosine kinase inhibitors; TWCs, total white cells.

filtered out by the software. A binomial test was applied to the remaining vari-ants to determine whether an appar-ent difference between samples could be due to inadequate read coverage in the control. Variants passing the bino-mial test were retained in the final list of putative somatic mutations (Fig. 2a) [8]. The identified mutations should be considered putative and, in com-mon with most other NGS strategies for the discovery of novel mutations, any specific mutation of clinical inter-est would need to be confirmed using

an independent method, as Sanger sequencing (limit of detection 20%), Sequenom MassArray, digital array (Fluidigm) or another NGS platform.

NGS: error rate, false positives and thresholdBefore the application of NGS technol-ogies, no evidence of heteroplasmy was detected, probably because of the lower sensitivity of earlier techniques [9]. NGS technologies enable the inquiry of mt heteroplasmy at the genome-wide scale with much higher resolution

because many independent reads are generated for each position [2]. How-ever, the higher error rate associated with the more sensitive NGS methodol-ogy must be taken into consideration to avoid false detection of heteroplasmy. Short-read sequencing technologies (like in Illumina systems) have a high intrinsic error rate (approximately 1 in 102–103 bases) when applied at the very high depth required to detect and measure low-level heteroplasmy. Thus, appropriate criteria for avoiding false positives due to sequencing errors are required. The most obvious way to dis-tinguish between sequencing errors and heteroplasmy is to invoke a threshold. Two duplicate sequencing run, of which one was ultra-deep (validation run), were compared to determine sensitivity (proportion of true positives that are correctly identified as such) and speci-ficity (proportion of true negatives that are correctly identified as such). An empirical threshold of 2% was there-fore applied to distinguish true variants from sequencing errors. Variants with a variant allele fraction (VAF, the vari-ant allele’s read depth divided by total read depth at each nucleotide position) between 2 and 98% where then con-sidered as heteroplasmic, and variants with a VAF >2% were called homoplas-mic [1]. This threshold could be refined by an iterative process in which a dif-ferent threshold is identified for each nucleotide position [10], as some vari-ation in error rate was observed. The incorporation of molecular barcodes in the initial long-range PCR would also reduce the risk of false-positive muta-tions due to PCR artefact [1].

Remission samples as control tissue in the identification of the mtDNA somatic mutations at diagnosisIn the four patients who had both MSC and hair follicle DNA available as con-trol tissue, the same mutations at diag-nosis have been identified, therefore the results using the non-hematopoietic tis-sues as control were combined. Remis-sion samples were then used as control tissue to determine mtDNA somatic mutations at diagnosis, and the con-cordance between this method and the conventional diagnosis versus the MSC/hair follicle approach was examined. Seventy-three somatic mutations (81%) were identified in common, 11 muta-tions (12%) were identified only in com-parison with the non-hematopoietic


Figure 2. (a) Schematic representation of the pipeline for mutation calling. For each test, diagnosis (Dx) and its matched control samples (remission or mesenchymal stromal cells-MSCs, hair follicles), LoFreq software was used to identify somatic mutations in the test sample based on comparison with the rCRS. Variants that were present in both test and matched control samples were filtered out, and an unfiltered list of putative somatic mutations was generated. Subsequently, an empirically determined VAF threshold of >2% was applied to reduce the risk of false discovery due to sequencing errors, leaving the final list of filtered putative somatic mutations in the test sample. (b) Venn diagram representing the agreement between mtDNA somatic mutations identified at diagnosis by using either remission samples (blue) or mesenchymal stromal cells/hair follicles (orange) as reference sequence. Dx, diagnosis; HF, hair follicles; MSCs, mesenchymal stromal cells; rCRS, revised Cambridge reference sequence; SNVs, single nucleotide variants; VAF, variant allele fraction.

control, and six (6.7%) only by compar-ison with remission samples (Fig. 2b) [1]. Divergent results occurred as the result of differences in read quality or depth at a specific nucleotide not reach-ing statistical significance in the algo-rithm. False-negative results could be encountered using remission samples as the control tissue, because of low-level heteroplasmic mutations in the con-trol sample that would lead to the same mutation at diagnosis being removed through filtering.

Concluding remarksRemission samples can be used as con-trol tissues to detect candidate mtDNA somatic mutations in leukemic samples when non-hematopoietic tissues are not available. The presence of muta-tions at low VAF in the remission sam-ples in common with the diagnosis tissue, could be filtered out by the LoF-req software leading to false-negative results. Therefore visual inspection of the unfiltered variants is recommended.

References1. Pagani IS, Kok CH, Saunders VA, van der Hoek MB, Heatley SL, Schwarer AP, Hahn CN, Hughes TP, White DL, Ross DM. A method for

next-generation sequencing of paired diagnos-tic and remission samples to detect mitochon-drial DNA mutations associated with leuke-mia. J Mol Diagn 2017; 19(5): 711–721.2. Li M, Schonberg A, Schaefer M, Schroeder R, Nasidze I, Stoneking M. Detecting hetero-plasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. Am J Hum Genet 2010; 87(2): 237–249.3. van Gisbergen MW, Voets AM, Starmans MH, de Coo IF, Yadak R, Hoffmann RF, Boutros PC, Smeets HJ, Dubois L, Lambin P. How do changes in the mtDNA and mitochon-drial dysfunction influence cancer and cancer therapy? Challenges, opportunities and mod-els. Mutat Res Rev Mutat Res 2015; 764: 16–30.4. Damm F, Bunke T, Thol F, Markus B, Wagner K, Gohring G, Schlegelberger B, Heil G, Reuter CW, et al. Prognostic implications and molec-ular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia. Leukemia 2012; 26(2): 289–295.5. Gattermann N. Mitochondrial DNA muta-tions in the hematopoietic system. Leukemia 2004; 18(1): 18–22.6. Yeung DT, Osborn MP, White DL, Branford S, Braley J, Herschtal A, Kornhauser M, Issa S, Hiwase DK, et al. TIDEL-II: first-line use of imatinib in CML with early switch to nilotinib for failure to achieve time-dependent molecu-lar targets. Blood 2015; 125(6): 915–923.

7. Meldrum C, Doyle MA, Tothill RW. Next-generation sequencing for cancer diagnostics: a practical perspective. Clin Biochem Rev 2011; 32(4): 177–195.8. Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, Chiea CK, Rosemary P, Martin LH, Niranjan N. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heteroge-neity from high-throughput sequencing datasets. Nucleic Acids Res 2012; 40(22): 11189–11201.9. Chatterjee A, Dasgupta S, Sidransky D. Mitochondrial subversion in cancer. Cancer Prev Res 2011; 4(5): 638–654.10. Kerpedjiev P, Frellsen J, Lindgreen S, Krogh A. Adaptable probabilistic mapping of short reads using position specific scoring matrices. BMC Bioinformatics 2014; 15: 100.

The authorIlaria Stefania Pagani1,2 PhD1Cancer Theme, South Australian Health & Medical Research Institute, Adelaide, Australia2School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia


– June 201823


DermatomycosisDermatomycoses are infections of the skin, hair and nails which are typically caused by dermatophytes and in rarer cases by yeasts and moulds. Fungal infections of the skin are the most frequently occurring infectious diseases globally with high and growing relapse rates. Elderly people and immunocompromised patients are especially at risk. Worldwide, around 20 to 25% of the popula-tion is affected by fungal skin diseases.

Infections which are caused exclusively by dermatophytes are referred to as dermatophytoses or tinea. Tinea pedis, which occurs

on the soles of the feet and between the toes, is one of the most fre-quent forms worldwide, followed by tinea unguium, which affects the nails, and tinea corporis, which affects the neck, back or trunk. Further forms, for example, on the face, legs, beard area, arms and hands, are rarer. Nail infections caused by dermatophytes and/or yeasts/moulds are called onychomycoses. They are typically accompanied by deformation of the nail.

Pathogens of dermatomycosisDermatophytes encompass fungi of the genera Trichophyton, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, Micro-sporum and Arthroderma. Individual species are classified as anthropophilic, zoophilic or geophilic according to their main occurrence. Human pathogenic yeasts and moulds include Can-dida spp., Scopulariopsis brevicaulis, Fusarium spp. and Aspergil-lus fumigatus.

Around 70% of human dermatophyte infections are caused by anthropophilic species. Trichophyton rubrum, in particular, is the most frequent cause of fungal skin infections worldwide. Infections can spread easily to other areas of the body or to other persons, for example, via showers, bathtubs or floors. Zoophilic dermatophytes are transmitted to humans by close contact with animals, especially pets, which are often asymptomatic. They can cause severe inflammatory reactions in humans. Geophilic derma-tophytes cause disease less frequently in humans. Infections typi-cally occur in gardeners and farm workers or children who play outside. Moulds and yeasts often cause opportunistic infections, benefitting from damage to the skin or nail caused by an exist-ing dermatophyte infection. In immunocompromised individuals, local fungal infections may develop into systemic mycosis.

Clinical pictureDermatomycoses are clinically heterogeneous and cannot always be differentiated from other dermatoses, such as eczema, psoriasis, erysipelas, or autoimmune diseases such as Lichen ruber planus. Furthermore, 5 to 15% of onychomycosis cases comprise mixed infections of dermatophytes with yeasts and moulds. Simultaneous


Direct detection and differentiation of dermatomycosis pathogens by DNA microarrayDermatomycoses are extremely widespread, and are characteristically long-lasting, recurring and very difficult to cure. Early and accurate identification of the causative agent is essential for targeted therapy. A new DNA microarray provides direct detection and differentiation of the most important dermatomycosis pathogens in one reaction. The assay simultaneously detects up to 50 dermatophyte species, and provides species identification for 23 of these, as well as 6 yeasts and moulds. The microarray analysis aids differential diagnosis of dermatomycoses from other dermatoses (e.g. psoriasis), and specifically identifies mixed infections with yeasts and moulds. The dermatomycosis microarray is part of the established EUROArray platform, which also includes microarrays for multiplex identification of sexually transmitted infections (STI) and complete detection and typing of human papillomaviruses (HPV).

by Dr Jacqueline Gosink

Table 1. Dermatophyte and yeast/mould species that are directly detected by the EUROArray.

Dermatophyte species

Anthropophilic T. simii

T. tonsurans T. quinckeanum* (T. mentagrophytes)

T. interdigitale T. erinacei

T. schoenleinii T. bullosum

T. concentricum T. benhamiae* (A. benhamiae)

T. rubrum T. verrucosum

T. violaceum T. eriotrephon

M. canis

M. ferrugineum N. persicolor* (M. persicolor)

M. audouinii Geophilic

Zoophilic N. fulva* (M. fulvum)

T. equinum N. gypsea* (M. gypseum)

T. mentagrophytes* (T. interdigitale ) N. incurvata* (M. incurvatum)

Yeast/mould species

C. parapsilosis F. solani

C. albicans F. oxysporum

C. guilliermondii Sc. brevicaulis

*new nomenclature (Hoog et al, Mycopathologia: 2017 Feb; 182(1-2):5-31)

bacterial infection of the damaged skin, pretreatment with corti-costeroid-containing preparations, or secondary contact allergy can also hinder diagnosis.

Dermatomycoses must always be treated. This is generally under-taken using various topical antifungal drugs, with severe cases sometimes requiring oral medication. Each drug has a limited activity spectrum. Positive pathogen identification prior to treat-ment enables targeted selection of the most suitable drug and opti-mal planning of the oftentimes lengthy therapy. In multiple infec-tions, a change of the primary pathogenic agent may occur during the therapy and it may seem like the therapy is failing. This must be taken into account in the treatment of fungal infections of the nails, which may only yield first success after months. Identifica-tion of the causative pathogen also helps to determine the source of the infection. In the case of zoophilic pathogens, for example, this is usually a pet.

Laboratory diagnosticsLaboratory diagnostic methods for identifying dermatomycosis pathogens include microscopic detection and an attempt at cul-turing from clinical material. Successful culture in most cases ena-bles species assignment based on micro- and macromorphological presentation of the fungus. Culturing is, however, time-consuming and is not possible for all dermatophytes. In mixed infections, false diagnoses may occur since slowly growing species may be over-grown or overlooked. Furthermore, antifungal therapy started before the sampling can hinder the culture.

Direct detection of pathogen genomic material by DNA micro-array enables secure and accurate identification of the causative agent. Microarray analysis offers a significant time advantage over detection by culturing, and is especially useful for detecting dermatophytes that are difficult to cultivate. It provides higher sensitivity and specificity, even in patients already undergoing treatment. The EUROArray Dermatomycosis analysis includes a universal dermatophyte detection encompassing 50 species of the genera Trichophyton, Epidermophyton, Microsporum, Nannizzia, Arthoderma, Lophophyton, as well as species identification for the 23 dermatophyte and 6 yeast and mould species listed in Table 1.

EUROArray procedureThe EUROArray procedure (Figure 1) is performed on DNA sam-ples isolated from skin scales, nail shavings or hair stubs. Defined gene sections of the pathogens are first amplified by multiplex polymerase chain reaction (PCR). The fluorescently labelled PCR products are then incubated with biochip microarray slides con-taining immobilized complementary probes. Specific binding (hybridization) of the PCR products to their corresponding oli-gonucleotide probes is detected using a special microarray scan-ner. The signals are evaluated and interpreted automatically by the EUROArrayScan software (Figure 2). A detailed result report is produced for each patient and all data are documented and archived. Meticulously designed primers and probes, ready-to-use PCR components and integrated controls all contribute to the reliability of the analysis. The entire EUROArray procedure from sample arrival to report release is IVD-validated and CE-regis-tered, supporting quality management in diagnostic laboratories.

Specifications and evaluationThe lower detection limit of the test system depends on the patho-gen and lies between 50 and 600 DNA copies per reaction, in indi-vidual cases also higher. Evaluation studies verified that template

DNA in concentrations ranging from the lower detection limit to 50 ng can be used in the PCR without generating any false posi-tive results. Furthermore, potential cross reactivity with 37 micro-organisms of the resident and transient skin flora was excluded experimentally.

In an evaluation study with 409 clinical samples, the EUROArray Dermatomycosis yielded a good agreement with the precharacter-ization. In many cases additional pathogens that were not included in the precharacterization were detected. The additional find-ings were confirmed by further independent tests or sequencing. Thus, the microarray provides reliable results and broad detection capabilities.

STI detectionThe EUROArray STI is based on the same technology and provides parallel direct detection of the pathogenic agents of eleven sexually transmitted infections (STIs) in one reaction, namely Chlamydia trachomatis, Neisseria gonorrhoea, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma par-vum, Haemophilus ducreyi, Treponema pallidum, Trichomonas vaginalis and herpes simplex viruses 1 and 2.

– June 201825

Figure 1. EUROArray procedure.

Figure 2. Evaluation of the EUROArray Dermatomycosis.

STIs are often asymptomatic, but can never-theless lead to serious sequelae, for example infertility, fetal damage during pregnancy, and severe postnatal infections in newborns. The PCR-based detection allows identifica-tion of both manifest and silent infections, and is thus suitable for diagnosis of sympto-matic patients as well as for general screen-ing. It offers a huge time advantage over cul-tivation and is especially useful for detecting sexually transmitted pathogens that are dif-ficult or impossible to cultivate, e.g. C. tra-chomatis, Mycoplasma, Ureaplasma, T. pal-lidum. Due to amplification of the pathogen

DNA, infections with a reduced pathogen number can also be reliably detected. A broad screening for STI pathogens is par-ticularly important in asymptomatic or clinically ambiguous cases and for detecting multiple infections, which are often missed during single-parameter testing.

HPV detection and typingThe EUROArray HPV provides detection and typing of all 30 genitally relevant HPV subtypes in one test. HPV are involved in the development of cervical carcinoma, and HPV testing plays a central role in risk

assessment and early diagnosis of this can-cer. In contrast to Pap examinations, HPV detection is not dependent on subjective evaluation and it offers very high sensitivity even in the early stages of infection. The EUROArray HPV is based on detection of the viral oncogenes E6 and E7, which pro-vides the highest possible sensitivity. Using an extensive panel of specific primers and probes, the EUROArray detects and distin-guishes between 18 high-risk subtypes that may trigger cancer (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and 12 low-risk subtypes that cause benign genital

warts (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89). Multiple infec-tions are reliably identified, and primary and persistent infections can be differentiated. A positive result for a high-risk subtype indicates an increased risk for cervical carcinoma, which can then be minimised by more fre-quent follow-up examinations to detect morphological cell changes at an early stage. Based on the recommendations of the respective professional societies, HPV-negative women can forgo subsequent HPV tests and Pap smears for a longer time interval.

ConclusionsMolecular diagnostic tests such as the EUROArray are an impor-tant tool for identifying the pre-cise pathogenic agent in various infectious diseases, supporting decision-making on specific treatment. The new EUROAr-ray Dermatomycosis provides the most comprehensive direct detection of dermatomycosis pathogens currently available commercially, and complements existing assays for STI and HPV. The EUROArray procedure is easy to perform and does not require extensive expertise in molecular biology. Moreover, the fully automated evaluation ensures objective, accurate and reproducible results. Further infectious disease microarrays based on the same technology are in development.

The authorJacqueline Gosink, PhDEUROIMMUN AG, Seekamp 31,23560 Luebeck, Germany

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A new drug to help young patients with genetic obesity

In a new study researchers from the Institute for Experimental Pediatric Endocrinology of the Charité – Universitätsmedizin Ber-lin have successfully treated patients whose obesity is caused by a genetic defect. Aside from its beneficial effects on the patients, the researchers also provided insights into the fundamental signalling pathways regu-lating satiety of the new drug. A mutation in the gene encoding the leptin receptor (LEPR) can cause extreme hunger starting with the first months of life. As a result, affected individuals develop extreme obesity during childhood. Increased exer-cise and reduced caloric intake are usually insufficient to stabilize body-weight. In many cases, obesity surgery fails to deliver any benefits, meaning that a drug-based treatment approach becomes increasingly important.Two years ago, Dr. Peter Kühnen and the working group successfully demonstrated that treatment with a peptide, which acti-vates the melanocortin 4 receptor (MC4R) could play a central role in the body’s energy metabolism and body weight regulation. Leptin, which is also known as the satiety (or starvation) hormone, normally binds to the LEPR, triggering a series of steps that leads to the production of melanocyte-stimulating hormone (MSH). The binding of MSH to its receptor, the melanocortin 4 receptor (MC4R) transduces the satiety sig-nal to the body. However, if LEPR is defec-tive, the signalling cascade is interrupted. The patient’s hunger remains unabated, placing her/him at greater risk of becom-ing obese. As part of this current study, researchers used a peptide that binds to the MC4R in the brain, and this activation triggers the normal satiety signal. Working in cooperation with the Clinical Research Unit at the Berlin Institute of Health (BIH), the researchers were able to record signifi-cant weight loss in patients with genetic defects affecting LEPR. “We also wanted to determine why the used peptide was so effective and why, in contrast to other preparations with a similar mode of action, it did not produce any severe side effects,” explains Dr. Kühnen. “We were able to demonstrate that this treatment leads to the activation of a specific and impor-tant signalling pathway, whose significance had previously been underestimated.” Dr. Kühnen’s team is planning to conduct fur-

ther research to determine whether other patients might benefit from this drug: “It is possible that other groups of patients with dysfunctions affecting the same signalling pathway might be suitable candidates for this treatment.”Charité – Universitätsmedizin Berlinhttps://tinyurl.com/y7dyaqm4

How epigenetic regulation of the Hoxb gene cluster maintains normal blood-forming cells and inhibits leukemia

Scientists have known for dec-ades that the Hox family of transcription factors are key regulators in the formation of blood cells

and the development of leukemia. Exactly how this large family of genes, which are distributed in four separate chromosomal clusters named A through D, is regulated has been less clear. Now, new research from the Stowers Institute for Medical Research reveals that a DNA regulatory element within the Hoxb cluster globally mediates signals to the majority of Hoxb genes to control their expression in blood-forming stem cells.“It’s like we found a general control that simultaneously turns the lights on and off in many rooms, rather than having a single switch that controls each individual room,” says Stowers Investigator Linheng Li, PhD, who co-led the study along with Stowers Scientific Director and Investigator Robb Krumlauf, PhD. These findings also help explain why a particular form of leukemia resists treatment and points to potential new therapeutic avenues.In mammals, the blood system contains a number of mature cell types — white blood cells, red blood cells, platelets — that arise from blood-forming, or hematopoietic, stem cells (HSCs). HSCs renew themselves and differentiate into other cells to replen-ish the body’s blood supply in a process called hematopoiesis. Hox genes, which are well known for their roles in establishing the body plan of developing organisms, are also important for HSCs to maintain their critical balancing act in the adult blood sys-tem, and have been implicated in the devel-opment of leukemia.In an article Li, Krumlauf, and co-authors including first author Pengxu Qian, PhD, second author Bony De Kumar, PhD, and other collaborators provide new details as

to how Hox genes are regulated in HSCs. They report that a single cis-regulatory ele-ment, DERARE, works over a long range to control the majority of Hoxb genes in HSCs in a coordinated manner. The research-ers found that the loss of the DERARE decreased Hoxb expression and altered the types of blood cells arising from HSCs, whereas “turning on” DERARE allowed Hoxb cluster gene expression in progeni-tor cells and increased the progression of leukemia.Genes can be regulated by non-coding DNA sequences termed cis-regulatory sequences. These sequences get input from multiple types of molecules, such as transcription factors, histone modifiers, or various morphogens. The DERARE, or distal element RARE (retinoic acid response element), is a cis-regulatory ele-ment that responds to signals from the vitamin A derivative retinoic acid and determines the fate of HSCs.Using human leukemia cell lines and mouse models, the Stowers researchers and col-laborators have identified a mechanism for how the retinoid-sensitive DERARE main-tains normal hematopoiesis and prevents acute myeloid leukemia (AML) by regulat-ing Hoxb cluster genes in a methylation-dependent manner.Methylation is the process of adding methyl groups to the DNA molecule, which can change the activity of the DNA segment. The researchers demonstrated that DNA methyltransferases mediate DNA meth-ylation on DERARE, leading to reduced Hoxb cluster expression. AML patients with mutations in the DNA methyltrans-ferase DNMT3A exhibit reduced DERARE methylation, elevated Hoxb expression, and adverse outcomes. “In two human AML cell lines carrying a DNMT3A mutation, we used an adapta-tion of genome editing technology called dCas9-DNMT3A to specifically increase the DNA methylation on DERARE. This targeted methylation technique was able to reduce Hoxb cluster expression and alleviate the progression of leukemia,” says Qian. “It is known that Hoxb cluster genes show a dramatic increase in expres-sion in patients with DNMT3A-mutated AML. Our work provides mechanistic insights into the use of DNA methylation on the DERARE as a potential screen-ing tool for therapeutic drugs that target DNMT3A-mutated AML, thus leading to the development of new drugs for treat-ing AML, in which DNA methylation is abnormal.”Stowers Institutehttps://tinyurl.com/yarf3xfd

NEWS IN BRIEF – June 2018 28

R-Biopharm Group enters into collaboration with SSI, Denmark in the field of tuberculosis diagnostics

R-Biopharm Group recently announced a collaborative agreement with SSI, focusing on discov-ery and develop-ment of novel diag-nostic approaches

for the detection of tuberculosis infection. The development of new diagnostic assays in the area of infectious diseases is a priority for R-Biopharm, a company with 30 years of experience in providing testing solutions for Clinical Diagnostics and Food & Feed analysis. R-Biopharm Group operates in vari-ous countries including subsidiaries in the UK, USA, Italy, France, Latin America, Brazil, Spain, Belgium, Australia, India and China as well as by a worldwide extensive network of more than 120 distributors. “This collaborative agreement creates an opportunity to work with the world-leading scientists and inventors of novel biomarkers and vaccines in the area of tuberculosis research. Our Infectious Dis-eases Team is inspired to join efforts for the development of novel diagnostics for the tuberculosis patients throughout the world. At R-Biopharm we are concerned about rising numbers of tubercu-losis cases and escalation of multidrug-resistant (MDR-TB) and extensively drug-resistant TB (XDR-TB). In cooperation with Statens Serum Institute we will have the potential to transform the current underserved market of tuberculosis diagnostics and pro-vide alternative solutions for the detection of TB infection,” said Dr. Ralf Dreher, CEO and Founder of R-Biopharm Group.SSI is one of Denmark’s largest research institutions in the health sector with over a century of experience in research, development and manufacturing of biologics. Led by Professor Peter Andersen, the research and development team at SSI are at the forefront in the development of novel vaccines and diagnostic agents for major diseases affecting global health. The program has brought several novel vaccine candidates in clinical trial within TB and Chlamydia as well as provided the ground work for the current industry standard in diagnostic tests for tuberculosis infection (IGRAs).“We are excited for our collaboration with R-Biopharm, a leading industry partner in the field of infectious disease diagnostics. As a government research organization under the ministry of health, industrial involvement is pivotal to get our projects from the lab out to the benefit of patients. The strong research base at R-Biop-harm is a great match for the SSI team and has been a fruitful syn-ergy,” said Prof. Peter Andersen, Executive Vice President, Center for Vaccine Research at Statens Serum Institute.Under the agreement, R-Biopharm and SSI will collaborate on research and development and responsibility for the commerciali-zation of potential products. www.r-biopharm.com

Siemens Healthineers, Hermes Pardini Group to create automated lab featuring the Atellica Solution Siemens Healthineers is developing “The Enterprise Project” with the Hermes Pardini Group of Minas Gerais, Brazil. The Enterprise Project is the largest and most complex clinical analysis laboratory known to date and is expected to be capable of handling 110 million sample tubes per year upon completion. Siemens Healthineers, in collaboration with Inpeco, has designed and will deliver this fully automated multidisciplinary solution on an unprecedented scale,

which will include at least 100 analysers—including more than 50 Atellica Solution clinical chemistry and immunoassay analysers from Siemens Healthineers, the largest IVD supplier in this pro-ject. The highly sophisticated solution will provide automation of clinical and operational workflow, from sample reception through testing to disposal. Sited in Vespasiano, Grande Belo Horizonte in Minas Gerais, the lab will occupy 3,500 square meters of floor space, will conduct operations 24 hours a day, and is expected to be fully operational during 2019. “The automation track will be more than 330 meters long upon completion and will be used to automatically transport and dis-tribute sample tubes to specific analysers that can run the spe-cific type of test requested by clinicians,” said Guilherme Collares, Chef Operations Officer of the Hermes Pardini Group. “Unlike conventional laboratory set-ups, where sample tubes have to be moved manually between different analysers, our enterprise lab is designed to employ a ‘one-touch, one workflow’ concept to eliminate the need for manual interventions, ensure sample trace-ability, and reduce the turnaround time to results. The Enterprise Project also will rely on Atellica Process Manager, an IT solution that delivers a 3D view of the laboratory configuration, to enable operators to manage alerts, control instruments and reagent mon-itoring remotely, and see test progression in real time. Siemens Healthineers will implement the first Laboratory Control Room, which will centralize management and provide holistic visibility of operations in the central Vespasiano laboratory, and other Hermes Pardini satellite lab units in São Paulo, Rio de Janeiro, Goiania, and Belo Horizonte.https://tinyurl.com/y9h4mtgm

– June 201829INDUSTRY NEWS

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PRODUCT HIGHLIGHT – June 2018 30

Introduction to the RX series The RX series combines robust hard-ware and intuitive software with a world leading test menu comprising of routine chemistries, specific pro-teins, lipids, therapeutic drugs, drugs of abuse, antioxidants and diabetes testing and a range of niche tests including the HbA1c assay. The RX series removes the need for a separate HbA1c analyser and allows laboratories to expand their test-ing capabilities onto one single platform, providing cost savings through consoli-dation. Built on three core values- reli-ability, accuracy and precision, the RX series reduces costly test re-runs and misdiagnosis, offering complete con-fidence in results. The RX series range of clinical chemistry analysers includes the RX misano, (semi-automated) RX monaco, RX daytona+, RX imola and RX modena.

BackgroundDiabetes is a global epidemic that affects approximately 271 million people around the world and according to the International Diabetes Federation; it is a figure that is on the rise. Their calcu-lations forecast that diabetes will affect roughly 552 million by the year 2030, highlighting the fundamental need to

manage patients with diabetes. The USA is one of the most prominent countries affected by diabetes when analysing its prevalence worldwide. It affects roughly 24 million Americans which is a stark contrast to the UK where around 3.5 million people have been diagnosed. It is also important to note that serious health complications can result from diabetes over time which reinforces the need to test HbA1c levels to evaluate how well diabetes is being controlled. The longer you have diabetes, the higher the risk of complications. These can include cardiovascular disease, nerve damage and kidney damage; includ-ing end-stage kidney disease which can require dialysis or a kidney transplant. With the rising figures and growing list of complications associated with the dis-ease, diabetes remains one of the lead-ing causes of death in the world and the seventh leading cause of death in the US.

HbA1c explained furtherHbA1c, also known as hemoglobin A1c or glycated hemoglobin, is an impor-tant blood test that is able to determine how well diabetes is being controlled. It develops when hemoglobin, a protein within red blood cells that carries oxy-gen throughout the body, joins with glu-

cose in the blood, becoming ‘glycated’. As glycation is irreversible, HbA1c remains in the same state in the red blood cell for 8-12 weeks; giving an overall picture of what the average blood sugar level is. This is particularly important as it allows clinicians to monitor the ‘glycemic’ con-trol in individuals with diabetes.

What is the HbA1c assay used for?The concentration of HbA1c in the blood of diabetic patients increases with rising blood glucose levels and is repre-sentative of the mean blood glucose level over the preceding six to eight weeks. HbA1c can therefore be described as a long term indicator of diabetic control unlike blood glucose which is only a short term indicator of diabetic control. It is recommended that HbA1c levels are monitored every three to four months. In patients who have recently changed their therapy or in those who have ges-tational diabetes it may be beneficial to measure HbA1c levels more frequently, at two to four week intervals. The onset of diabetes, particularly type two diabe-tes, tends to be gradual and thus proves difficult to diagnose early. With HbA1c clinicians are able to monitor blood glu-cose levels periodically to deliver accu-rate and quick results that can improve patient care.

The clinical significance of HbA1c The measurement of HbA1c is used in the long term monitoring of diabetes mellitus. This assay should not be used in the diagnosis of diabetes mellitus or for day to day glucose monitoring. Diabetes

Direct HbA1c testing capabilities on the RX series range of clinical chemistry analysersThe global prevalence of diabetes mellitus is increasing rapidly; affecting 8.8% of the population. The Randox automated immunoturbidimetric HbA1c test offers an improved method for the rapid direct measurement of HbA1c in human blood; which is available for use on the RX modena, RX imola and the RX daytona+.

RX daytona+ RX imola RX modena

– June 201831

mellitus is a disease associated with poor glycemic control. Numerous clinical studies, including the Diabetes Control and Complications Trial, have shown that diabetes-related com-plications may be reduced by the long term monitoring and tight control of blood glucose levels. In the diabetic patient where blood glucose levels are abnormally elevated the level of HbA1c also increases, the reason for this is that HbA1c is formed by the non-enzymatic glycation of the N-terminus of the ß-chain of hemoglobin A0.

Randox HbA1c assay features:• Sample type – suitable for use with whole blood samples• Latex enhanced immunoassay method – the Randox

assay utilizes an immunoassay method making it simple and quick to perform.

• Liquid ready to use reagents – for ease of use and convenience

• Excellent stability – all reagents are stable to expiry date when stored at +2-8ºC or 28 days on board the analyser at approximately 10°C.

Advantages of the RX series clinical chemistry analysers for direct HbA1c testing:

• Fully automated on-board haemolysis function for HbA1c testing

• Continuous loading & STAT sample functionality to enhance productivity in the laboratory- analyser dependant

• Low sample volumes required• 1200 tests per hour including ISE (RX modena)• User friendly software• Low water consumption• Dual 5 speed stirrers optimized for reach chemistry

reaction • Reagent micropipette with liquid level sensor and crash

detection • Liquid level sensor, crash, bubble and clot detection

Key benefits of using the RX series clinical chemistry analysers and Randox HbA1c assay Direct HbA1c testing eliminates the need for the sample incu-bation step which is required on alternative methods; allow-ing samples to be to run immediately on the RX series and providing faster and more accurate results when they are needed. The removal of the offline preparation stage increases the recovery times, allowing laboratories to enhance their workflow and also consolidate testing onto one single clinical chemistry platform. Additional benefits of using the RX series in conjunction with the Randox HbA1c assay include having one assay instead of two, which enables quicker calibration; saving the user time and making the overall method simple and quick to perform, setting a new standard in HbA1c deter-mination and patient care.

For further information or for a quotation for the HbA1c test or to enquire about any analyser in the RX series range, please contact [email protected].

Randox Laboratories Ltd55 Diamond RdCrumlin, Co. Antrim BT29 4QYUK

www.randox.com

Contact us for more information : [email protected] DIA

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European CE marking has been confirmed for Beckman Coulter’s Early Sepsis Indicator,

a hematology-based solution designed to alert emergency department clinicians to the possibility of sepsis or risk of develop-ing sepsis. The first early sepsis warning solution to be offered as part of a routine CBC with differential test, the Early Sep-sis Indicator gives physicians a rapid and simple tool that can aid in the fight against sepsis. The new marker will be commer-cially available on the recently launched DxH 900 hematology analyser. Sepsis is an often-deadly condition that affects 26 million people worldwide every year and is increasing at a rate of 1.5% annually. Timely and accurate detection solutions in the acute-care setting are key compo-nents to stopping the progression of sep-sis, as patients with less severe sepsis can progress to severe sepsis or septic shock within 72 hours. Up to half of patients with sepsis die. In addition to the human toll, this global crisis places a significant clini-cal and economic burden on the health-care system. A clear link exists between the timeliness of treatment and the possibility of death. When antibiotics are adminis-tered early to patients with septic shock,

the likelihood of death is decreased by 7.6% per hour. Because emergency depart-ment personnel are often on the front line of care for people facing critical condi-tions, giving them a simple and easy tool for detecting sepsis can help make signifi-cant strides against this prevailing threat. The fact that this early warning indicator is part of a routine blood test means that clinicians receive results rapidly, with no additional workflow burden to the labo-ratory or emergency department. The Early Sepsis Indicator uses the DxH 900 hematology analyser’s Coulter technology, which characterizes cells in their near-native states. The system’s powerful VCS 360 technology can uniquely detect mor-phological changes in monocytes - cells of the innate immune system that provide a first line of defense against infections. Monocytes play a role in the dysregulated immune response to sepsis, and identifying morphological changes provides insight into possible sepsis earlier than other indi-cators. The company plans to submit a 510(k) for the Early Sepsis Indicator to the U.S. FDA in the near future. After receiving 510(k) clearance, the Early Sepsis Indicator will be released to the U.S. market.

BECKMAN COULTER DIAGNOSTICS AACC Booth 3612 www.clinlabint.com & search 27717

Automated ESR testing instrument

CUBE 30 Touch is an automated instrument for high-volume erythrocyte sedimentation rate (ESR) testing in EDTA tubes. Com-patible with stand-ard 4.0 mL K2EDTA

tubes, the instrument produces ESR results directly from EDTA tubes without consum-ing patient sample. Its internal mixing func-tion automatically prepares up to 30 samples per batch. Random access capability enables the user to add samples as space allows. The instrument automatically prints and trans-mits results to the laboratory information system (LIS). Results are available in under 25 minutes and show >94% correlation to Modified Westergren Method. An internal barcode scanner ensures positive patient ID. Patient and QC data are archived and a USB port and Bluetooth connector allows con-nection to PC, tablet and smartphone. No reagents are needed and there is no exposure to patient sample. Utilizing plastic tubes, the instrument does not produce any liquid waste, thus saving costs for waste disposal.

DIESSE AACC Booth 4422 www.clinlabint.com & search 27719

PRODUCT NEWS – June 2018 32

Beckman Coulter recently announced the release of the DxH 900 hematology analyser, giving mid- to high-volume clinical laboratories the ability to per-form complete blood count (CBC) and white blood cell differential tests with minimal repeats. The DxH 900 is now available for sale in Europe, the United States, Canada, Australia and New

Zealand. The DxH 900 analyser offers advanced technolo-gies to support patient care, by delivering the right results the first time. Foundational to the system are its core technolo-gies, including the enhanced Coulter Principle, VCS 360 and DataFusion. These features offer high-resolution analysis of cells in their near-native states, providing a precise cellular assessment for excellent red blood cell, platelet and white blood cell test results on the first pass. The suite of technolo-gies is intended to help laboratories deliver quality results for fast, accurate clinical decision-making. At the same time, the system includes automated solutions that streamline the number of procedural steps needed to produce those results, offering predictable performance and greater laboratory effi-ciency. The DxH 900 analyser demonstrates 93% first-pass

throughput, providing accurate flagging and reducing the number of slide reviews. This helps to generate reportable results as quickly as possible, reducing the time, supplies and costs that may be required for systems with higher repeat rates. Adding to this is the analyser’s lean reagent portfolio, which includes four reagents compared to eleven reagents required by other analysers. Further, the DxH 900 features one of the smallest footprints in its class, making it highly efficient in utilization of laboratory space. Many of the parameters available with the DxH 900 analyser are designed to directly impact patient care by addressing critical conditions, such as thrombocytopenia, anemia and leukopenia. Beckman Coul-ter is evaluating a hematology sepsis parameter that is part of a routinely ordered test in the emergency department, where earlier recognition and treatment of sepsis can begin. This test can be performed on the DxH 900 analyser (see below). The DxH 900 hematology analyser complements the recently announced DxH 520 system, and further expands Beckman Coulter’s hematology portfolio.

BECKMAN COULTER DIAGNOSTICSAACC Booth 3612 www.clinlabint.com & search 27716

Hematology analyser provides near native-state cellular characterization

Beckman Coulter achieves CE Mark for its Early Sepsis Indicator

Hemoglobin and hematocrit meter systemThe latest addition to the StatStrip line of hand-held, hospital meters, the StatStrip Hemoglobin and Hematocrit Meter System (StatStrip Hb/Hct), has gained CE mark certification and is now available in all CE regulated countries. StatStrip Hb/Hct is the only point-of-care (POC) meter to measure hemoglobin and hematocrit for accurate anemia screening and blood loss monitoring. StatStrip Hb/Hct provides measured hemoglobin

and hematocrit results, which are more accurate than calculated results, with excellent correlation to laboratory reference methods. With a fin-gerstick capillary sample and results in 40 seconds, StatStrip Hb/Hct provides the real-time accuracy that helps improve clinical decision making in a variety of healthcare settings. These include blood banks and temporary blood collection locations, dialysis centres, primary care clinics, hospitals, and oncology clinics. For example, in blood banks, StatStrip Hb/Hct provides safe and effective blood donor screening and avoids false deferrals. For patients receiving dialysis, StatStrip Hb/Hct helps maintain Hb target levels and also monitor erythropoiesis stim-ulating agents (ESAs) to the lowest effective dose. In emergency care settings and in hospitals, StatStrip Hb/Hct can aid in rapidly evaluat-ing blood loss, initiating treatments more quickly, and monitoring criti-cally ill patients who are at risk for low hemoglobin and hematocrit. In oncology clinics, StatStrip Hb/Hct can help proactively identify patents at high risk for chemotherapy-induced anemia and direct treatment accordingly. StatStrip Hb/Hct’s single-use biosensors do not require cal-ibration or coding, and testing is as easy as glucose self-testing. StatStrip Hb/Hct helps reduce costs by eliminating the need for blood drawing supplies, venous phlebotomy, and laboratory testing. Wired and wire-less connectivity for data integration with patient records are available. Compact and lightweight, StatStrip Hb/Hct is less than half the size and weight of other POC systems that measure only hemoglobin.

NOVA BIOMEDICALAACC Booth 1814 www.clinlabint.com & search 27720

Reagent kit for mAb drugs by LC-MS/MS Shimadzu’s new nSMOL Antibody BA reagent kit offers a unique and stand-ardized workflow for the quantita-tion of monoclonal antibody drugs by LC-MS/MS in blood or other biological samples. The technique, called nano-Surface and Molecular-Orientation

Limited (nSMOL) proteolysis, has been designed to minimize sam-ple complexity while maintaining the specificity of the protein sub-strate for LC–MS/MS quantitation in therapeutic drug monitoring of mAbs. nSMOL proteolysis is an entirely novel solid-solid prote-olysis method engineered to deliver highly efficient and quantitative detection of CDR (Complementarity-Determining Region) pep-tides while decreasing the peptide numbers of the analytical target without antibody denaturation.

SHIMADZU AACC Booth 4478 www.clinlabint.com & search 62027

– June 201833PRODUCT NEWS

Free 25OH Vitamin D kit is a CE-marked ELISA kit available worldwide from Diasource ImmunoAssays SA (Belgium). Recent posters and publi-cations clearly show that Free 25OH Vitamin D is a better marker than Total 25OH Vitamin D in healthy

pregnant women, in women taking hormonal contraceptives and in diabetic patients with impaired kidney function. This kit allows a direct measurement of the Free 25OH Vitamin D, so no calculations are needed based on Total 25OH Vita-min D -, Albumin - and VDBP (vitamin D binding protein) concentrations, and this without sample pre-treatment. The assay is calibrated against Rate Dialysis, which is the golden standard method for the measurement of free hormones, and uses only 10 microliters of serum.

DIASOURCE IMMUNOASSAYS AACC Booth 3642 www.clinlabint.com & search 27702


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Identification of OXA-23 carbap-enem-resistant Acinetobacter spp.

The global spread of carbapenem-resistant Acine-tobacter spp., and particularly A. bau-mannii, in health-care settings causes

a major threat to patient survival. World-wide, drug-resistant Acinetobacter spp. are responsible for serious nosocomial out-breaks, mostly in intensive care units. Even though different resistance mechanisms have been described, production of Amber Class D oxacillinases OXA-23-like hydroly-sis enzymes is the most frequently acquired mechanism. Fast and accurate detection of OXA-23-producing strains remains chal-lenging at the laboratory level. Specific iden-tification of oxacillinase-expressing Acineto-bacter requires genotypic molecular assays that remain expensive and not commer-cially available. However immunochroma-tography lateral flow tests are cost-effective and convenient to detect drug-hydrolysing enzymes. Today, a large panel of lateral flow tests to detect carbapenemase-expressing Enterobacteriacea, named “RESIST”, is com-mercialized by the Belgian company Coris BioConcept. In order to fulfill the unmet need for fast and reliable detection of drug-resistant Acinetobacter, Coris BioConcept developed a new lateral flow assay, “OXA-23 K-SeT”, for the specific identification of OXA-23-mediated carbapenem-resistant

in Acinetobacter spp. from a single colony. With results available in less than 15 min-utes, this easy-to-use and instrument-free rapid test represents the first phenotypic, specific and non-molecular confirmatory assay for OXA-23 detection. It will expand the RESIST range of products already adopted nowadays by routine and reference laboratories worldwide, offering an addi-tional tool to ensure the successful treat-ment of patients and to prevent the spread of carbapenemase-producing bacteria in healthcare settings.

CORIS BIOCONCEPTAACC Booth 3742


Valveless 400 μl dispensing pump

Fluid Metering, Inc.(FMI) intro-duces their new 400 μl dispensing pump ideal for medical, analytical and biotech Instru-

mentation. Having the identical compact design dimensions as their STH & STF OEM pump lines, the STF1-9 expands dispense and metering capabilities of previous STH designs by 100% while maintaining 0.5% precision. The STF1-9 is available in nine drive configurations

ranging from 200 μl through 400 μl in 50 μl increments. Each drive model (STF1, STF2 … STF9) has an adjustable displace-ment of ±25 μl. A supplied adjustment tool rotates an eccentric bushing to precisely make micro-volume adjustments. There are also four standard pump head options available to provide a fluid path with max-imum chemical and dispense volume/flow rate compatibility. Fluid Metering’s STF1-9 utilizes FMI’s CeramPump valve-less fluid transfer technology. One mov-ing part accomplishes both the pumping and valveless functions within the pump, thereby eliminating valves present in other reciprocating pump designs. Sap-phire-hard ceramic internal components are both chemically inert and dimension-ally stable, resulting in a pump that will transfer fluid, in micro-volume amounts, at a precision of 0.5% or better for mil-lions of maintenance-free cycles. FLUID METERING AACC Booth 2142 www.clinlabint.com & search 27697

PRODUCT NEWS – June 2018 34

STA Compact Max 3, a new addition to the line of Max G e n e r a t i o n hemostasis sys-tems, is a multi-

parameter automated analyser that offers some important new functionalities. Its EPC (expert preanalytical check) mod-ule allows full control of fill volumes and detection of hemolysed, icteric and lipemic (HIL) samples directly at the nee-dle without consuming plasma and with-out affecting throughput. This new module has improved the reliability of its viscos-ity-based detection system (VBDS), the proven standardized chronometric tech-nology present throughout the range. In its category, the STA Compact Max 3 ana-lyser has further optimized productivity

by keeping manual maintenance opera-tions to a minimum and improving ergo-nomics, thanks to technological advances in its pipetting system including a new PSR module (Single Resolution Pipettor) and new cap piercing needle. New soft-ware tools offer more effective emergency management (STAT) to optimize TAT (turnaround time) and improve trace-ability. The analyser has increased its field of expertise, allowing full management of reflex testing rules based on renewed clinical algorithms. Quality control has also improved thanks to these develop-ments and includes an externalized iQC web platform – My Expert QC – which can connect to the analyser.

STAGOAACC Booth 1224www.clinlabint.com & search 27718


CALENDAR OF EVENTS

July 29-Aug 2, 2018AACCChicago, IL, USAwww.aacc.org

Sept 9-12, 201830th European Con-gress of Pathology Bilbao, Spainwww.esp-congress.org

Sept 9-13, 2018MSACL 2018 EUSalzburg, Austriawww.msacl.org

Sept 23-26, 201821st ESCVAthens, Greecewww.escv2018.com

Sept 30-Oct 2, 2018BSACIBritish Society for Allergy & Clinical Immu-nologyTelford, UKwww.bsacimeet-ing.org

Oct 2-4, 2018Medlab EuropeBarcelona, Spainwww.medlabeu-rope.com/en/home.html

Oct 29-Nov 1, 2018CMEF AutumnShanghai, Chinawww.cmef.com.cn

Dates and descriptions of future events have been obtained from official industrial sources. CLi cannot be held responsible for

errors, changes or cancellations.

For more events see:www.clinlabint.com/events/

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*Product availability will vary by country. †Dependent on test mix. ‡Versus leading IVD companies.

Atellica is a trademark of Siemens Healthcare Diagnostics Inc. A91DX-9564-A2-4A00. © Siemens Healthcare Diagnostics Inc., 2017

Experience the power of theAtellica Solution

Atellica® Solution:* Flexible, scalable, automation-ready immunoassay and chemistry analyzers engineered to deliver control and simplicity so you can drive be�er outcomes.

A bi-directional magnetic sample transport that is 10 times faster than conventional sample conveyors

Unprecedented flexibility with more than 300 customizable configurations including L and U shapes

An immunoassay analyzer that runs up to 440 tests per hour,† the industry’s highest productivity per square meter‡

The new standard in sample management—revolutionary technology that gives independent control over every sample

Visit us at AACC

Booth #2812


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