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C347 Paolis Figure1 v2 new crop

Competitive PCR-high resolution melting analysis: an improved approach to assess BRCA status in hereditary breast and ovarian cancer patients

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Widespread use of BRCA molecular testing has been observed in recent decades relating to the approval of PARP inhibitor as a target therapy for breast and ovarian cancer in BRCA-positive patients. This article provides an overview of the crucial issues of the BRCA test, focusing on our innovative cPCR-HRMA technology.
by Elisa De Paolis, Dr Angelo Minucci, Dr Giovanni Luca Scaglione, Maria De Bonis and Prof. Ettore Capoluongo
The relevance of BRCA analysis
The identification of BRCA pathogenic variants (PVs) is the major concern for the genetic counselling in families with a high risk of breast (BC) and ovarian cancer (OC). BRCA1 (breast cancer early onset 1) and BRCA2 (breast cancer early onset 2) are the two major susceptibility genes in BC/OC, conferring a lifetime risk up to 87% for BC and up to 44% for OC. BRCA mutations have been found in 4–14% of all OC, with a higher occurrence of about 22% in the high-grade serous OC [1]. The clinical relevance of the identification of BRCA PV carriers concerns many aspects of a patient’s evaluation. The first relevant implication is the assessment of the lifetime cancer risk. Additionally, BRCA testing has a relevant impact on the therapeutic approach and on the treatment outcomes owing to the possibility of selecting patients for biomarker-directed therapy based on the mutational status [2]. BRCA-positive patients with OC, particularly, respond well to platinum-based chemotherapy, especially in the high-grade serous OC subtype, and tend to retain platinum-sensitivity for longer than those without BRCA PVs. Additionally, the treatment with poly (ADP-ribose) polymerase (PARP) inhibitor (e.g. olaparib) was approved as a target therapy in patients with both germline and somatic BRCA PVs. PARP inhibitor therapy is able to improve progression-free survival in response to a recent platinum-based chemotherapy [3]. To date, licensed PARP inhibitor is part of the standard care and, consequently, BRCA evaluation is considered a routine investigation tool, useful before treatment management. With respect to these benefits, BRCA testing should be offered to all patients with OC on the basis of histological subtype, regardless of age, or family and personal history of malignancy. This issue causes an increase of the demand for BRCA testing with a strong challenge into the diagnostic laboratories committed in fulfilling the need of an efficient and rapid molecular evaluation [4].

The challenge of BRCA testing
To date 1700 PVs in BRCA1 and 1900 PVs in BRCA2 have been reported. Most of them are single nucleotide polymorphisms (SNPs) or small insertion-deletion mutations (indels), with a significant impact on the structure and function of the protein. Also large genomic rearrangements (LGRs), consisting mainly in large deletions or duplications, represent an important part of BRCA molecular lesions. To date, a total of 98 different BRCA LGRs have been reported, 81 in BRCA1 and 17 in BRCA2 [5] with a prevalence that varies considerably. Interestingly, deletion of BRCA1 exon 1a-2 is reported in several populations worldwide and is considered a recurrent BRCA LGRs in BC/OC patients [6]. Owing to the broad complexity in the mutational landscape of BRCA genes, comprehensive screening including the efficient assessment of both qualitative (SNPs, indels) and quantitative (LGRs) alterations is mandatory (Fig. 1). Diagnostic laboratories are adopting next-generation sequencing (NGS) technology for BRCA testing, which offers the potential of fast, cost-efficient and comprehensive sequencing. By choosing NGS technology, many considerations should be made, such as the selection of an NGS platform, including the enrichment methods, the sequencing chemistries, the analytical procedures and the variant calling for both germline and somatic PVs [2]. NGS is highly recommended as the reference sequencing method for BRCA testing because of the size of coding region and the method’s sensitivity in tumour sample evaluation. In fact, Sanger sequencing is not suitable for the analysis of somatic mutations, especially in samples where the percentage of tumour cells is under 50%, and it requires also a large amount of starting DNA [4]. Several methods are commonly used for LGR analysis, including multiplex ligation-dependent probe amplification (MLPA) and multiplex amplicon quantification (MAQ). However, these approaches are expensive and time-consuming, and consequently these are not always suitable for all laboratories. In this case, LGR evaluation of BRCA genes represents a bottleneck in terms of time and costs. In this context, the great benefit of the NGS approach is the opportunity to obtain both qualitative and quantitative information from the same sequencing data by using tailored bioinformatics algorithms [7]. Only a positive bioinformatics result needs to be confirmed using an alternative conventional method. In order to optimize our routine diagnostic procedures for BRCA testing, we recently developed a new molecular approach called competitive PCR-high resolution melting analysis (cPCR-HRMA) [8], as an alternative method for LGR identification in BRCA genes. HRMA is a simple and robust closed-tube method commonly used for diagnostics, forensic and research purposes. This method consists of a PCR amplification performed in the presence of saturating binding dyes followed by a melting reaction. Specifically, the incremental increase of the reaction temperature causes the denaturation of double-stranded DNA with the concomitant release of intercalated dye and a decrease of fluorescence signal. The specific sequence of the analysed amplicon, primarily relating to the GC content and the length, determines the melting behaviour observed in a fluorescence signal versus temperature plot. Additionally, the melting temperature (Tm) may be calculated as the derivative of the melting curve. The shape of the curves and the specific Tm value obtained in the output plots is used for the genotyping. The advantages of this technique include rapid turn-around times and a closed-system environment that decrease the risk of laboratory contamination [9].

cPCR-HRMA for LGR evaluation
HRMA technology is typically applied to detect a single substitution, as well as small indels variants [6, 10]. The new cPCR-HRMA represents an optimized HRMA method that allows an efficient evaluation of BRCA1 copy number variation (CNV) by relying on the melting behaviour of target BRCA amplicon compared to a reference amplicon in the same HRMA reaction. In particular, specific albumin sequences were chosen as unchanged CNV references and analysed by coupling them with specific BRCA1 exons in a duplex PCR assay preceding the melting analyses. The landmarks of this new HRMA rely on the primers and the amplification protocol design. First of all, primer pairs for the simultaneous amplification of target and reference sequences are selected in order to produce paired amplicons with comparable lengths (similar amplification efficiencies) and different melting temperatures (no overlap between amplicons melting peaks). Furthermore, the primer concentration used for both target and reference amplification was set in order to produce comparable PCR performance between the two amplicon types and to obtain melting profiles more suggestive of CNV. In addition, the PCR thermal cycling was carried on until the exponential phase, in which the amplification performance reflects the CNV status of the target region. These optimized features lead to melting profiles specifically tailored for CNV investigations allowing a rapid detection of samples affected by a change in copy number. Genotype association was assessed by direct interpretation of melting profiles, as shown in Figure 2: samples with similar profiles were clustered into the same genotype group and CNV positive samples showed a typical melting profile with a detectable shape comparing to the wild type. In addition to the qualitative evaluation, we provide also a semi-quantitative analysis of melting behaviour with the calculation of the fluorescence peak height ratio (R) of target the amplicon (BRCA1) to the reference amplicon (albumin), according to the formula:
The mean and the standard deviation of the R values calculated in a consistent number of control CNV samples allowed the identification of the reference range for the R parameter: WT sample (mean±2SD; 2 copies), deletion (≤mean−2SD; n copies) and duplication (≥mean+2SD; 3n copies). The R value calculated in each analysed sample is normalized with the average of the ratios calculated in the WT sample group, obtaining the normalized fluorescence peak height ratio (Rn). The latter is compared to the reference range in order to obtain the copy number interpretation. Taken together, the qualitative and the semi-quantitative evaluations of the cPCR-HRMA assay allow the correct identification of copy number status in BRCA gene, resulting as a rapid and alternative method for the analysis of LGRs. Advantages of cPCR-HRMA are the ease and fast handling of samples. Furthermore, this application needs the same reagents and equipment for standard HRMA protocols commonly used in many laboratories routines. By introducing this efficient alternative method, our first aim was the optimization of the BRCA workflow, promoting a more rational use of confirmatory testing, such as MLPA and MAQ. Finally, we are confident that a general implementation of BRCA testing is now necessary as an emerging challenge. After a complete genetic counselling and a multidisciplinary activity that involves geneticists, oncologist and all other professionals, the patient should be directed to specialized laboratories. The complexity of the potential BRCA mutations, coupled with their clinical relevance, leads to the mandatory adoption of a comprehensive molecular workflow for BRCA analysis that must be characterized by a low wait-time and efficient clinical reporting in order to guarantee a useful medical application.

References
1. Antoniou A, Pharoah PD, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003; 72(5): 1117–1130.
2. Capoluongo E, Ellison G, López-Guerreroc JA, et al. Guidance statement on BRCA1/2 tumor testing in ovarian cancer patients. Semin Oncol 2017; 44(3): 187–197.
3. George A, Kaye S, Banerjee S. Delivering widespread BRCA testing and PARP inhibition to patients with ovarian cancer. Nat Rev Clin Oncol 2017; 14(5): 284–296.
4. Capoluongo E, Scambia G, Nabholtz JM. Main implications related to the switch to BRCA1/2 tumor testing in ovarian cancer patients: a proposal of a consensus. Oncotarget 2018; 9(28): 19463–19468.
5. Sluiter MD, van Rensburg EJ. Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation. Breast Cancer Res Treat 2011; 125: 325–349.
6. Mazoyer S. Genomic rearrangements in the BRCA1 and BRCA2 genes. Hum Mutat 2005; 25(5): 415–422.
7. Scaglione GL, Concolino P, De Bonis M, et al. A whole germline BRCA2 gene deletion: how to learn from CNV in silico analysis. Int J Mol Sci 2018; 19(4): pii: E961.
8. Minucci A, De Paolis E, Concolino P, et al. Competitive PCR-high resolution melting analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: a new approach to assess quantitative status of BRCA1 gene in a reference laboratory. Clin Chim Acta 2017; 470: 83–92.
9. Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 2008; 85(1): 50–58.
10. De Paolis E, Minucci A, De Bonis M,  et al. A rapid screening of a recurrent CYP24A1 pathogenic variant opens the way to molecular testing for idiopathic infantile hypercalcemia (IIH). Clin Chim Acta. (2018) Mar 21; 482: 8–13.

The authors
Elisa De Paolis MSc, Angelo Minucci PhD, Giovanni Luca Scaglione PhD, Maria De Bonis MSc, Ettore Capoluongo* PhD
Catholic University of The Sacred Heart, Rome, Italy
*Corresponding author
E-mail: ettoredomenico.capoluongo@policlinicogemelli.it

C344 Dorwal Fig 1 crop

Ber-EP4 (CD326) testing by flow cytometry: a rationalized algorithm-based approach

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Flow cytometry has traditionally been used to identify hemato-lymphoid neoplasms. However, the flow cytometry laboratories that deal with tissues would often receive samples that have an epithelial neoplasm. In our laboratory, we use flow cytometry to identify cells with epithelial differentiation using Ber-EP4 antibody that targets CD326 (EpCAM). We have formulated an algorithm-based approach for the application of this marker. This approach has been elaborated in this article.

by Dr Pranav Dorwal and Dr Helen Moore

Introduction
The use of flow cytometry in the laboratory has traditionally been applied for diagnosing lymphomas and leukemias. The biggest advantage that flow cytometry has over histopathology is a much quicker turn-around-time, as most of the samples are fresh and can be processed right away, unlike a histopathology sample which needs to undergo fixation and processing before it is ready to be examined. Any additional testing on flow cytometry samples can be performed instantly, whereas the same usually requires another day in the histopathology lab. Many of the lymph node malignancies (primary lymphomas versus metastatic involvement) can appear undifferentiated. In these cases, the histopathologist needs the help of a plethora of immunohistochemical markers to reach a diagnosis. The ability to identify samples where non-hematological malignancies are present can be helpful for the treating physician as well as the reporting histopathologist, who can then test with a more dedicated panel. The ultimate aim of this testing is to get an early diagnosis so that patient’s treatment is not delayed.

A large number of markers have been used to identify epithelial differentiation in tumours by immunohistochemistry (IHC), including cytokeratin (CK), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125) as well as epitopes recognized by the antibodies LeuM1 (anti-CD15 antibody), and MOC-31 and Ber-EP4 antibodies, both of which recognize epitopes on EpCAM (the epithelial cell adhesion molecule). However, most of these are not available for use by flow cytometry. EpCAM (also known as CD326) was first discovered in 1979 and at that time thought to be specific for colonic carcinoma [1]. Ber-EP4 is, therefore, an anti-CD326 antibody which binds to a cell membrane glycoprotein on human epithelia. There is a comprehensive list of tumours that are Ber-EP4 positive, as described by Went et al. and Spizzo et al. [2, 3]. The traditional use of Ber-EP4 in histopathology has been limited essentially for differentiation between adenocarcinoma and malignant mesothelioma [4]. This could be due to the fact that other epithelial markers (such as CK) are expressed more often than the CD326 (EpCAM) in epithelial malignancies and thus are more helpful in lineage determination.

We use an algorithmic approach to decide the flow cytometry panel to be applied (Fig. 1). When the clinical details or radiological findings are indicative of a non-hematopoietic malignancy, we apply the CD326 panel. This panel is composed of CD326, CD56 and CD45. CD56 was included in the panel to identify myeloma cells (which may be present in the CD45-negative region) and cells with neuroendocrine differentiation. If, on analysis, there is no CD45-negative population and the sample is composed of predominantly lymphoid cells, a lymphoid screening panel is then used. Samples that are received with diagnosis of suspected lymphoma are initially processed with a routine lymphoid screening panel. In these cases, Ber-EP4 antibody is tested only if large numbers of CD45-negative events are identified.

Method for Ber-EP4 testing
The tissue and fine-needle aspirate (FNA) samples are received fresh in RPMI medium. The tissues are placed on the metal sieve and ground using a glass pestle to form a cell suspension using 2 % PBS-FCS. This suspension is subsequently filtered, which is then washed and lysed. The cell count is ascertained by the cell counter only in cases of larger tissues, where we may have to dilute the sample to adjust the cell count to approximately 10×109/L. FNA and core biopsies are usually paucicellular and do not need a cell count.

The sample is stained with 5 µl of CD45-PC5 [Immunotech SAS (Beckman Coulter)], 20 µl of CD56-PE (Immunotech SAS) and 10 µl of monoclonal mouse anti-human epithelial antigen-FITC conjugated antibody (Clone: Ber-EP4) (Dako Denmark A/S). The sample is then incubated at 4 °C for 30 minutes, followed by a washing step and is ready to be run on the flow cytometer (Beckman Coulter Life Sciences). A total of 10 000 events are acquired with the time threshold set at 300 seconds for the acquisition.

Flow cytometric analysis
The flow cytometric analysis is performed using Navios and Kaluza softwares (Beckman Coulter Life Sciences). The various populations of interest are gated with the focus on identifying the expression of CD326 (with or without CD56) in the CD45-negative population.

Discussion
In our experience of testing for CD326 by flow cytometry, we have been able to comment on the presence or absence of CD326 expression in CD45-negative populations (Figs 2(a, b) and 3). The various carcinomas where we have identified CD326 positivity are: adenocarcinoma, small cell carcinoma, Merkel cell carcinoma, renal cell carcinoma, squamous cell carcinoma, prostate carcinoma, germ cell tumour of testis, and myxoma. We have observed that the expression of CD326 in melanomas can be variable, but they more frequently express CD56. The co-expression of CD326 and CD56 usually indicates a neuroendocrine tumour. Our concordance rate with histopathology using CD326 testing was found to be 97.6 %, which we have published previously [5].

CD326 expression has also been reported to be a prognostic marker with poor outcomes in epithelial ovarian and gall bladder carcinomas [6, 7]. Another important role of this testing could be application in decision making for use of monoclonal antibodies for targeted therapy. The first EpCAM targeting antibody, Catumaxomab (trade name Removab, Fresenius Biotech GmbH) received European market approval in EpCAM-positive carcinomas for the treatment of malignant ascites. Another modification that could be useful in diagnosing epithelial malignancies is to apply Ki67 testing using flow cytometry. This could be done in the same tube as CD326, and thus more information could be obtained with the same amount of sample [8].

There has been considerable data describing the use of the Ber-EP4 antibody in malignant effusions [9–11]. The literature mentions that the presence of epithelial cells in the body fluid should raise the suspicion of metastatic epithelial malignancy, as the reactive body fluids may be composed of lymphocytes and reactive mesothelial cells in varying proportions. There have been multiple studies in the past where flow cytometric CD326 testing has been applied for identifying epithelial cells in body fluid effusions. We have found that our results have a very good concordance with histopathology results. This is in keeping with the findings of Davidson et al., although their study looked at the detection of malignant cells in effusions [12].

The disadvantage of using Ber-EP4 for identifying epithelial differentiation is that there are many epithelial malignancies that do not express CD326 (EpCAM). As mentioned earlier, the use of a broader antibody like cytokeratin (pan-CK) may solve this problem. But unfortunately, such an antibody is not currently available for clinical use by flow cytometry, to the best of our knowledge. Meanwhile, Ber-EP4 should give us the answer in most of the cases. Another disadvantage is that CD326 will be negative in cases of neoplasms of mesenchymal origin, such as sarcomas.
Most flow cytometry laboratories across the world will liaise with histopathology departments for the diagnosis of non-Hodgkin lymphomas. The use of Ber-EP4-testing flow cytometry may play an important role even in epithelial malignancies. The antibody used by us is a CE-marked antibody for in vitro diagnostics and, thus, requires a limited verification process. We followed the method recommended by the manufacturer. The rapid turn-around-time of flow cytometry results makes it a useful screening tool. Our experience shows that flow cytometric testing for CD326 (EpCAM) can be a useful method for diagnosing non-lymphoid malignancies that are poorly differentiated. We suggest that this method would be more useful if the protocol for its application is set up in consultation with the histopathology department, along with setting up a channel of bilateral communication. The histopathologist, based on the flow cytometry information provided, can then set up a more directed immunohistochemical panel. We would like to emphasize at this stage that the aim of the flow cytometric CD326 testing is not to formally diagnose carcinomas, but to highlight the presence of epithelial cells which may lead to the diagnosis of carcinoma. Final classification obviously remains the role of the histopathologist.

References
1. Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 2012; 38(1): 68–75.
2. Went PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum Pathol 2004; 35(1): 122–128.
3. Spizzo G, Fong D, Wurm M, Ensinger C, Obrist P, Hofer C, Mazzoleni G, Gastl G, Went P. EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J Clin Pathol 2011; 64(5): 415–420.
4. Sheibani K, Shin SS, Kezirian J, Weiss LM. Ber-EP4 antibody as a discriminant in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. Am J Surg Pathol 1991; 15(8): 779–784.
5. Dorwal P, Moore H, Stewart P, Harrison B, Monaghan J. CD326 (EpCAM) testing by flow cytometric BerEP4 antibody is a useful and rapid adjunct to histopathology. Cytometry B Clin Cytom 2017; doi: 10.1002/cyto.b.21543.
6. Spizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol 2006; 103(2): 483–488.
7. Varga M, Obrist P, Schneeberger S, Mühlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Schäfer G, et al. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res 2004; 10(9): 3131–3136.
8. Sikora J, Dworacki G, Zeromski J. DNA ploidy, S-phase, and Ki-67 antigen expression in the evaluation of cell content of pleural effusions. Lung 1996; 174: 303-313.
9. Pillai V, Cibas ES, Dorfman DM. A simplified flow cytometric immunophenotyping procedure for the diagnosis of effusions caused by epithelial malignancies. A J Clin Pathol 2013; 139(5): 672–681.
10. Krishan A, Ganjei‐Azar P, Hamelik R, Sharma D, Reis I, Nadji M. Flow immunocytochemistry of marker expression in cells from body cavity fluids. Cytometry A 2010; 77(2): 132–143.
11. Risberg B, Davidson B, Dong HP, Nesland JM, Berner A. Flow cytometric immunophenotyping of serous effusions and peritoneal washings: comparison with immunocytochemistry and morphological findings. J Clin Pathol 2000; 53(7): 513–517.
12. Davidson B, Dong HP, Berner A, Christensen J, Nielsen S, Johansen P, Bryne M, Asschenfeldt P, Risberg B. Detection of malignant epithelial cells in effusions using flow cytometric immunophenotyping. Am J Clin Pathol 2002; 118(1): 85–92.

The authors
Pranav Dorwal* MBBS, DCP, DNB; Helen Moore MBChB, FRACP, FRCPA
Waikato Hospital, Pembroke St, Hamilton 3204, New Zealand

*Corresponding author
E-mail: Pranav.dorwal@waikatodhb.health.nz

C351 Wood BCBM Risk Factors

Risk factors for development of breast cancer bone metastasis

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Breast 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

Introduction
Invasive 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 metastatic 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 complications 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 treatment 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 postmenopausal 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 targeted to those who will benefit, sparing those who will not.

Risk factors
Clinicopathological and demographic risk factors
Breast cancer is a heterogeneous disease and pathological staging and grading systems are widely used in routine practice. Although not generally specific for indicating risk of bone metastasis, these systems do categorize patients into sub-groups that determine appropriate treatment 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 expression of genes including the progesterone receptor (PR). Patients with HER2-positive breast cancer have a poorer prognosis, but targeted treatments are now available. 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-negative patients. Although these pathological categories are routinely examined, there has been a recent strong research emphasis upon the discovery of molecular risk factors for development of metastasis, including BCBM.
Molecular risk factors for bone metastasis
Genetic risk factors
There is good evidence that the risk of breast cancer spread to bone can be predicted 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 metastasize 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 expression of multiple genes, can influence the probability of breast cancer spread to different target tissues.

Genes that predict BCBM have been discovered using de novo unbiased genetic screening approaches – including gene copy-number 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 immunocompromised 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 treatment are not observed in patients with MAF-positive tumours [12].

Breast cancer cells which have metastasized to bone frequently remain dormant for many years as disseminated tumour cells (DTCs). Growth signals that are still not completely understood trigger eventual 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 stratify patients in terms of risk of developing BCBM.

Protein-expression risk factors within BCBM
Several studies have focused on altered protein expression within BCBM. Immunohistochemical 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 subsequent development of BCBM [14]. The absence of cytoplasmic OPN in this study was observed to be an independent risk factor for the development of BCBM, whereas expression of PTHR1 was observed to be associated with BCBM; however, the association was not significant within multivariate analysis, thus PTHr1 levels are not an independent predictor of BCBM [14].

Quantitative proteomic analysis of parental 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 protein (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 suggesting that these two markers are also predictive of treatment benefit.

Bone morphogenetic protein-7 (BMP7) is a cytokine which can elicit diverse signalling outcomes within breast cancer cells, including altering the rates of cell migration, invasion 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 development of BCBM within invasive ductal carcinomas [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 mineralized 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 development of BCBM
Bone turnover markers are products of active bone resorption and formation. Several of these markers are products of collagen metabolism including procollagen-I N-terminal extension pro-peptide (PINP) and procollagen-I C-terminal extension peptide (PICP) – markers of bone formation, as well as C-terminal type-I collagen telopeptide (CTX) and C-terminal telopeptide (ICTP) – markers of bone resorption [21]. In a study measuring the levels 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 treatment benefit from ZA [22].

In a related study [23], Lipton et al. investigated 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 significant 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-terminal telopeptide of collagen (NTX), PINP and tartrate resistant acid phosphatase (TRACP5b; a marker of bone resorption), were measured in postmenopausal women with early stage luminal-type invasive 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].
Conclusion
The 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 profiling 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, differential 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 intrinsic 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 signalling and cell migration) that may form the basis of future tests.

Once within bone, breast cancer cells trigger alterations in the bone micro-environment that favour survival of DTCs. Later when macroscopic metastases form, the altered rates of bone formation and breakdown lead to the generation of bone metabolic products that can be measured within patients. Altered levels of these bone metabolic products predict BCBM development and can also be used to monitor treatment responses. Extracellular matrix components 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 (ranging from animal model systems to patient-derived samples), as well as variations in the patient cohorts used for different clinical 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 candidate molecules in larger, multicentre clinical trials will further refine a testing panel for prediction of BCBM risk.

References
1. Cancer Research UK (CRUK). Breast cancer statistics (http: //www.cancerresearchuk.org/health-professional/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 features, pathophysiology and treatment strategies. Cancer 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 preventing 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, Spensley 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. Patterns of breast cancer relapse in accordance to biological 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 expression 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 international, 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, Salvador F, Urosevic J, Figueras-Puig C, Rojo F, Del Barco Barrantes I, et al. MSK1 regulates luminal cell differentiation 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. Immunohistochemical 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 cancer 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, Savinainen K, Karhu R, Kallioniemi A. BMP7 influences proliferation, migration, and invasion of breast cancer cells. Cancer Lett 2009; 275(1): 35–43.
17. Alarmo EL, Korhonen T, Kuukasjarvi T, Huhtala H, Holli K, Kallioniemi A. Bone morphogenetic protein 7 expression associates with bone metastasis in breast carcinomas. Ann Oncol 2008; 19(2): 308–314.
18. Bahrami A, Hassanian SM, Khazaei M, Hasanzadeh M, Shahidsales S, Maftouh M, Ferns GA, Avan A. The therapeutic potential of targeting tumor microenvironment 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 revascularization after marrow ablation is dramatically accelerated in BSP-/- mice, along with faster hematopoietic recolonization. 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 multivariate logistic regression analysis of accuracy. Clin Chim Acta 2016; 460: 227–230.

The authors
Steven L. Wood MA, PhD; Prof. Janet E. Brown* BMedSci, MB BS, MSc, MD, FRCP
Academic Unit of Clinical Oncology, Department of Oncology and Metabolism,
University of Sheffield, UK

*Corresponding author
E-mail: j.e.brown@sheffield.ac.uk

Scientific Lit picture 09

Scientific literature review: Pathology

, /in /by
An assessment of the effect of haemoglobin variants on detection by faecal immunochemical tests
Carroll MR, John C, Mantio D, Djedovic NK, Benton SC. Ann Clin Biochem 2018; doi: 10.1177/0004563218778716 [Epub ahead of print]
BACKGROUND: Faecal immunochemical tests (FIT) for haemoglobin (Hb) are being used in the investigation of colorectal cancer. These tests use antibodies raised to the globin moiety of human Hb. Where the globin structure is abnormal or reduced, it is possible that antibody binding, and thus Hb-detection may be affected.
METHODS: Lysates prepared from whole blood samples of patients with known variants were diluted in manufacturer-specific buffer to 10, 100 and 500 μg Hb/g feces. These samples were analysed on four FIT analysers and the results compared with samples with no known variant present (normal samples).
RESULTS: The results from this study show that of 20 variants tested, three showed a decrease in detection by all four analysers. These were β-thalassemia major and two fetal cord blood samples.
CONCLUSIONS: Of 20 common Hb variants studied, 17 did not affect detection of Hb by the FIT systems tested. Hb variants leading to a reduction in the presence of a globin chain caused a reduction in Hb detection; in such cases, cancers could be missed.
Thirty-three-day storage of dithiothreitol-treated red blood cells used to eliminate daratumumab interference in serological testing
Lorenzen H, Lone Akhtar N, Nielsen M, Svendsen L, Andersen P. Vox Sang 2018; doi: 10.1111/vox.12699 [Epub ahead of print]
BACKGROUND AND OBJECTIVES: Daratumumab binds CD38 on red blood cells causing interference with indirect antiglobulin tests. Dithiothreitol is used to eliminate interference allowing detection of alloantibodies. Hemolysis is observed during storage of dithiothreitol-treated antibody identification panel cells. The objective of this study was to develop a modified method for dithiothreitol treatment to reduce the hemolysis during 33 days of storage and still be able to eliminate daratumumab interference.
MATERIALS AND METHODS: Panel cells were treated with various volumes of 0·2 m dithiothreitol supplied by various manufacturers. Hemolysis Index of dithiothreitol-treated and untreated panel cells was measured and compared on days 1, 15 and 33. Antibody screening tests with dithiothreitol-treated screening cells were performed on samples from 15 daratumumab-treated patients (dose 16 mg/kg) and 34 patients with known alloantibodies. Antibody identifications with dithiothreitol-treated panel cells were performed on seven additional known alloantibodies.
RESULTS: Dithiothreitol treatment with a ratio of 30:25 (red blood cells:dithiothreitol) showed the same degree of hemolysis as with untreated panel cells. Daratumumab interference was eliminated in all 15 samples from daratumumab-treated patients. Twenty-six of 34 alloantibodies were detected, and all seven additional alloantibodies were identified using the modified dithiothreitol treatment. Eight alloantibodies within the Kell system were negative. No decrease in the reaction strength was observed during the 33-day storage period.
CONCLUSION: The modified dithiothreitol method was able to reduce hemolysis during storage and to detect and identify alloantibodies in the presence of daratumumab.
The assessment of iodine status – populations, individuals and limitations
Wainwright P, Cook P. Ann Clin Biochem 2018; doi: 10.1177/0004563218774816 [Epub ahead of print]
Iodine deficiency is a significant global health concern, and the single greatest cause of preventable cognitive impairment. It is also a growing public health concern in the UK particularly among pregnant women. Biomarkers such as urinary iodine concentration have clear utility in epidemiological studies to investigate population-level iodine status, but determination of iodine status in individuals is much more problematic with current assays. This article reviews the available biomarkers of iodine status and their relative utility at the level of both populations and individuals for the investigation of iodine deficiency and iodine excess.
How low can you go? Analytical performance of five automated testosterone immunoassays
La’ulu SL, Kalp KJ, Straseski JA. Clin Biochem 2018; 58: 64–71
BACKGROUND: Testosterone is commonly measured using immunoassays, yet concerns with the accuracy and quality of testing by these methods exist, particularly for low testosterone concentrations. Study objectives were to evaluate selective performance characteristics, including functional sensitivity (FS), of five automated immunoassays for total testosterone.
METHODS: FS, imprecision, assay interference, limit of blank, linearity, and accuracy were assessed using the Abbott ARCHITECT i2000SR, SIEMENS ADVIA Centaur and IMMULITE 2000, Beckman Coulter DxI 800, and Roche MODULAR E170. Comparisons to an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were performed using patient samples from men, women, boys, and girls.
RESULTS: FS at 20% coefficient of variation (CV) for the ARCHITECT, Centaur, DxI, E170 and IMMULITE assays were 0.14, 1.23, 0.36, 0.77, 3.49 nmol/L, respectively. Total CVs for the 5-day imprecision study were ≤9.0% for all methods. All assays met manufacturer’s claims for hemolysis, icterus, and lipemia interference and limit of blank. Dilution linearity studies had deviations from the target recoveries ranging from 3.4% (ARCHITECT) to 14.3% (DxI). Using National Institute of Standards and Technology Standard Reference Material 971, recoveries ranged from 79.2–149.2% (DxI, male and female, respectively). When compared to LC-MS/MS, more immunoassays under-recovered in men and women and over-recovered in boys and girls. Slopes ranged from 0.71 (IMMULITE, women) to 1.35 (DxI, boys). The combined average for percent bias was higher in boys (28.0%) than men (11.6%), women (22.8%), and girls (25.7%).
CONCLUSIONS: Challenges with accurately measuring testosterone appear to remain for some immunoassays, but not all. While most immunoassays remain optimized for concentrations observed in healthy men, some showed acceptable performance when challenged at lower concentrations.
27651 DiaSys Anzeige CLI Qdx VitD 92x132 eng 180305 PRINT

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The new nSMOL Antibody Bioanalysis Kit

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C339 Boknas Fig 1 flow chart

Establishing flow cytometry as a primary diagnostic method for the investigation of suspected platelet function disorders

, /in /by

Although considerable progress has been made in our understanding of the role of platelets in hemostasis, the analytical methods clinically available for investigating platelet function defects remain limited. Herein, we describe an initiative at Linköping University Hospital, Sweden, to use flow cytometry for measuring platelet function in patients with a suspected bleeding disorder.

by Dr Niklas Boknäs, Dr Sofia Ramström and Prof. Tomas Lindahl

Introduction
Although many patients seek professional help for bleeding problems, very few end up receiving an informative diagnosis, even when the presenting symptoms are clearly abnormal [1]. At present, our diagnostic tools for the investigation of bleeding symptoms are tailored for identifying serious disorders with dramatic symptoms such as hemophilia and Glanzmann’s thrombastenia, but often fail to identify the underlying defect in mild bleeding disorders (MBD) [2]. Ironically, the reverse is also often true, as the clinical significance of many tests performed during conventional laboratory investigations of MBDs is ill-defined [3].

Platelet function disorders (PFDs) represent a subcategory of MBDs where the underlying hemostatic defect is caused by abnormally low platelet pro-hemostatic activity. As PFDs produce virtually identical clinical symptoms to many other conditions causing bleeding problems, diagnosing PFDs necessitates access to reliable laboratory testing of platelet function. Ideally, such tests could provide important guidance in a number of clinical situations, such as when deciding on whether to give pharmaceutical prophylaxis in the event of frequent bleeding or surgery and when assessing the risks associated with the use of thromboprophylaxis after thrombosis and surgery in the individual patient.

Unfortunately, clinical tests evaluating platelet function have evolved poorly during recent decades, despite the introduction of new promising techniques. Light transmission aggregometry (LTA), the method currently considered gold standard for evaluating platelet function, has been used for more than five decades and comprises continuous measurement of the optical density of stirred platelet-rich plasma after stimulation with agonists. LTA gives information about how platelets aggregate upon stimulation, but does not enable measurement of other aspects of platelet pro-hemostatic activity such as platelet adhesion, granule secretion and alterations of platelet membrane structure to accelerate coagulation. From our experience, the clinical value of LTA in terms of explaining patient symptoms is limited, and this is supported by studies failing to show an association between results from LTA and the severity of bleeding problems among patients with MBD [1, 4]. In addition to this limitation, LTA remains poorly standardized and labour-intensive, making performance of LTA only feasible in specialized hemostasis laboratories.

Flow cytometry for the diagnosis of PFD in patients with MBD
In an effort to overcome these problems with the methods currently used for diagnosing PFD, we and others have switched to employing whole-blood flow cytometry for the diagnosis of PFD among patients with MBD. Whole-blood flow cytometry for platelet function testing (FC-PFT) was developed in the 1980s [5, 6]. A description of the analytical principle behind flow cytometry is outside the scope of this article, but in this context, the technique can extremely briefly be described as a powerful method to quantify the presence of different epitopes on the surface of platelets after platelet activation by the use of fluorescent probes that bind to the cell surface. Compared to LTA, FC-PFT confers the following practical advantages [7]:

  • Samples can be analysed in anticoagulated whole blood, eliminating the need for pre-analytical manipulation of blood components.
  • Sample volumes can be reduced drastically, which is especially advantageous in children.
  • Results are not influenced by platelet count, enabling assessment of platelet function in patients with thrombocytopenia [8].
  • The work load is reduced considerably as many samples can be analysed in rapid sequence.
  • Flow cytometry is a very common technique, and appropriate instruments are widely available in most clinical and research laboratories.

In addition to these practical benefits with FC-PFT, the method confers several other advantages. For example, it produces numerical results that are easy to interpret, and can give information about several different aspects of platelet activation by the employment of different fluorescent probes detecting distinct events during platelet activation [9]. The ability to measure different aspects of platelet function also allows the direct diagnosis of rare disorders, such as Bernard-Soulier syndrome, Glanzmann’s thrombastenia and Scott syndrome, without the need for sequential testing [10].

Unfortunately, until recently no studies had addressed the clinical utility of FC-PFT for diagnosing clinically relevant PFDs. To address this issue, we recently published a clinical study comparing the results from FC-PFT with symptom severity in a cohort of bleeders [11]. The study was performed on 105 patients referred to Linköping University for evaluation of platelet function. Only patients wherein a complete diagnostic work-up including a full blood cell count, APTT (activated partial thromboplastin time), PT (prothrombin time), FVIII (factor 8) and von Willebrand factor (antigen and ristocetin cofactor activity) had excluded the presence of von Willebrand disease or a coagulation disorder were included in the study. Bleeding symptoms were assessed by a single experienced clinician blinded to the laboratory results of the study. In our panel for FC-PFT, we included analysis of fibrinogen binding (indicating activation of the fibrinogen receptor glycoprotein (GP)IIb/IIIa responsible for platelet aggregations) as well as P-selectin exposure (indicating release of platelet alpha granules) after platelet stimulation with a panel of four different agonists that specifically activate the most important platelet receptors: P2Y12 and P2Y1 (ADP); the thrombin receptors PAR1 and PAR4 [PAR1-activating peptide (AP), PAR4-AP]; and the collagen receptor GPVI (CRP-XL). To assess the contribution of dense granules to platelet activation, we designed an indirect test wherein the effects of pre-incubation with apyrase (which degrades ADP) was used as a measure of functional dense granule release. A flow chart illustrating the flow cytometry protocol is provided in Figure 1.

Our results clearly demonstrate that abnormal test results using FC-PFT are associated with a more severe bleeding phenotype in patients with MBDs. In fact, a high symptom burden was 5–8 times more common among patients with more than two abnormal test results in our study as compared to patients with two or fewer abnormal test results (Fig. 2), depending on which method that was used for calculating the reference range for the different tests. When results pertaining to the fifth percentile of the patient material was classified as abnormal and more than two abnormal test results were used as a predictor for bleeding symptom severity, a high symptom burden was predicted with as specificity of 95 % and a positive predictive value of 80 %. It should be noted however, that the clinical material was insufficient to allow for a prospective validation of these estimates in a separate patient cohort.

Discussion
In our opinion, FC-PFT for clinical use should as a minimum comprise: (a) testing of platelet integrin activation, either directly by the use of the anti-PAC-1 antibody (recognizing GPIIb/IIIa) or indirectly by measuring fibrinogen binding or microaggregate formation; (b) a marker of alpha granule secretion, preferably by using an antibody directed towards P-selectin; and (c) a test of dense granule secretion to accurately assess the clinically most important hemostatic functions of platelets. Ideally, a clinical protocol for FC-PFT should also include a marker of platelet procoagulant platelet activity and a fluorescent marker binding to GPIbα, in order to provide a more complete assessment of the platelet hemostatic repertoire and diagnose the rare hereditary disorders Scott syndrome and Bernard-Soulier syndrome. In our own protocol, we have recently incorporated these two additional functionalities. We have also improved our protocol by incorporating the use of fixatives and pre-preparation of frozen reagents in order to improve reproducibility and increase the time- and cost-efficiency of the protocol. Recently, very promising methodological improvements have been made by other researchers, such as the use of fluorescent beads as an internal control for standardizing results and facilitating comparisons between different instruments [12] and the use of a modular diagnostic algorithm to ensure efficient and exact diagnosis [13]. Thus, continuous efforts are being made to firmly establish FC-PFT as an attractive alternative for platelet function testing in the setting of MBDs.

References
1. Quiroga T, Goycoolea M, Panes O, Aranda E, Martínez C, Belmont S, Muñoz B, Zúñiga P, Pereira J, Mezzano D. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007; 92(3): 357–365.
2. Quiroga T, Mezzano D. Is my patient a bleeder? A diagnostic framework for mild bleeding disorders. ASH Educ Progr B 2012; 2012(1): 466–474.
3. Harrison P. Platelet function analysis. Blood Rev 2005; 19(2): 111–123.
4. Lowe GC, Lordkipanidzé M, Watson SP, UK GAPP study group. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013; 11(9): 1663–1668.
5. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70(1): 307–315.
6. Lindahl TL, Festin R, Larsson A. Studies of fibrinogen binding to platelets by flow cytometry: an improved method for studies of platelet activation. Thromb Haemost 1992; 68(2): 221–225.
7. Michelson A. Flow cytometry: a clinical test of platelet function. Blood 1996; 87: 4925–4936.
8. Frelinger AL, 3rd, Grace RF, Gerrits AJ, Berny-Lang MA, Brown T, Carmichael SL, Neufeld EJ, Michelson AD. Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP. Blood 2015; 126(7): 873–880.
9. Ramström S, Södergren AL, Tynngård N, Lindahl TL. Platelet function determined by flow cytometry: new perspectives? Semin Thromb Hemost 2016; 42(3): 268–281.
10. Rubak P, Nissen PH, Kristensen SD, Hvas A-M. Investigation of platelet function and platelet disorders using flow cytometry. Platelets 2015; 27(1): 66–74.
11. Boknäs N, Ramström S, Faxälv L, Lindahl TL. Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders. Platelets 2017; doi: https://doi.org/10.1080/09537104.2017.1349305
12. Huskens D, Sang Y, Konings J, van der Vorm L, de Laat B, Kelchtermans H, Roest M. Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test. PLoS One 2018; 13(2): 1–16.
13. Andres O, Henning K, Strauß G, Pflug A, Manukjan G, Schulze H. Diagnosis of platelet function disorders: a standardized, rational, and modular flow cytometric approach. Platelets 2017; doi: 10.1080/09537104.2017.1386297.

The authors
Niklas Boknäs*1,2 MD, PhD; Sofia Ramström3,4 PhD; Tomas Lindahl3 MD, PhD
1Department of Hematology and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
2Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
3Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
4School of Medical Sciences, Örebro University, Örebro, Sweden

*Corresponding author
E-mail: niklas.boknas@gmail.com

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