In your hands, this sample has the power to change the direction of a life. What you do with this sample can provide critical insights after an organ transplant that a doctor, a patient, a family, will count on.
Together let’s bring exceptional quality to molecular diagnostic testing.
For more information on Bio-Rad’s molecular quality controls for transplant visit: info.bio-rad.com/Molecular-Transplant-2020
by Dr Weijie Li
Cytogenetic and molecular testing has been becoming more and more important in the diagnosis of human diseases. These tests are indispensable for the diagnosis and/or prognostic prediction for some hematopoietic malignancies. This article discusses a group of lymphomas with similar morphology and phenotype but distinct cytogenetic/molecular abnormalities, with emphasis on the importance of proper cytogenetic/molecular testing in the diagnosis of these diseases.
Burkitt lymphoma (BL) is an aggressive high-grade B-cell lymphoma (HGBCL), which typically shows sheets of monomorphic mediumsized tumour cells with round nuclei, finely dispersed chromatin, and multiple basophilic small to intermediate-sized nucleoli (Fig. 1a). The cytoplasm is moderately abundant and highly basophilic with multiple lipid vacuoles better visualized on Wright’s and/or Giemsa stained air-dried smears or imprint slides. There are many mitotic and apoptotic figures, and numerous intermixed tingible body macrophages resulting in a so-called ‘starry sky’ pattern. The immunophenotype of typical BL is that of germinal centre B-cell-type (GCB), positive for immunoglobulin (Ig) M, CD19, CD20, CD22, and CD79a, CD10 and BCL6 (see Table 1 for definitions). BCL2 is usually negative. The proliferative index (as determined by Ki-67 levels) is nearly 100% (Fig. 1a) .
There are three clinical variants of BL: endemic, sporadic and immunodeficiency-associated. They differ mainly in their geographic distribution, clinical presentation and anatomic localization of primary tumour. Regardless of the clinical variants, the molecular hallmark of BL is the translocation of the MYC proto-oncogene (MYC) (8q24) to the Ig heavy chain region (14q32), t(8;14) (q24;q32), or less commonly to the Ig light chain kappa locus on 2p12 or the Ig light chain lambda locus on 22q11. There are other molecular cytogenetic changes, which include copy-number gains involving 1q, 7 and 12 and losses involving 6q, 13q32-34 and 17p. Gene expression profiling studies have defined a molecular signature characteristic of BL, which is different from other lymphomas such as diffuse large B-cell lymphoma (DLBCL) . Mutations of the transcription factor 3 (TCF3) gene or the gene for its negative regulator, inhibitor of DNA binding 3 (ID3) have been frequently detected by next-generation sequencing analysis in sporadic BL cases. The resulting mutant proteins can activate B-cell receptor signalling, which sustains BL cell survival by engaging the phosphoinositide- 3-kinase pathway. Other mutations in CCND3, TP53, RHOA, SMARCA4 and ARID1A genes are also detected in certain BLs. Endemic cases show fewer mutations overall and lower proportion of mutations in TCF3 or ID3 .
Technically, all BL cases should have MYC rearrangement. It is controversial to make a diagnosis of BL when MYC rearrangement is not detectable. However, routine fluorescence in situ hybridization (FISH) and chromosomal analysis may not be able to detect this translocation owing to complex karyotype or more complicated translocation. Additionally, by gene expression profile study, some MYC-negative cases show a typical BL molecular signature . Therefore, in the presence of classic morphology and phenotype, the diagnosis of BL should be made even without the demonstration of this translocation. Recent studies [3, 4] show that some true MYC-negative cases have characteristic 11q aberration, which has been recognized by the 2016 WHO classification as a provisional new entity [1, 5] and will be discussed more in detail next.
Historically, there has been no consensus on the diagnostic classification of the lymphomas with morphology resembling BL but showing more cytological pleomorphism and/or other atypical morphologic or phenotypic features. These were so-called ‘grey-zone lymphomas’ and classified as atypical BL/Burkitt-like lymphoma (BLL) by the 2001 WHO Classification of tumours of hematopoietic and lymphoid tissues. The name was then changed to B-cell lymphoma, unclassifiable, with features intermediate between BL and DLBCL by 2008 WHO classification . These lymphomas were thought to represent a continuum between BL and DLBCL.
2016 revision of the WHO classification
The 2016 WHO classification of tumours of hematopoietic and lymphoid tissues has shed some light on this field with the modification of the grey-zone lymphoma with features intermediate between BL and DLBCL, and the creation of Burkitt-like lymphoma with 11q aberration (BLL-11q) and large B-cell lymphoma with IRF4 rearrangement (LBL-IRF4) [1, 5].
HGBCL with MYC and BCL2 and/or BCL6 rearrangement and HGBCL, not otherwise specified
The grey-zone lymphoma with features intermediate between BL and DLBCL has been divided into two categories: HGBCL with MYC and BCL2 and/or BCL6 rearrangement (double-hit or triple-hit lymphomas); and HGBCL, not otherwise specified (NOS). These lymphomas usually occur in older adults, with men and women affected equally. They are usually present as a rapidly enlarging mass involving lymph nodes or extranodal sites with advanced stage. These lymphomas have very poor prognosis with no optimal therapeutic strategy [7–9]. Double-hit (DH)/triple-hit (TH) refers to the co-occurrence of MYC and BCL2 and/or BCL6 translocations. MYC rearrangements usually differ from those seen in BL: they often involve non-IG partner genes. These lymphomas usually have complex karyotypes. The majority (60–70%) of them have MYC/BCL2 rearrangements, with a minority (15–30%) harbouring MYC/BCL6 rearrangements and fewer (10%) harbouring rearrangements of all three genes. Rare cases of follicular lymphoma or B-lymphoblastic leukemia/lymphoma can have these DH/TH cytogenetic changes. They should not be included in this category. HGBCLs with DH/TH morphologically can resemble BL, DLBCL or lymphoblastic lymphoma. They are phenotypically mature B-cell lymphomas with the presence of CD19, CD20, CD79a, and Pax-5 and lack of TdT proteins. Most cases also produce CD10 and BCL6. HGBCL, NOS includes cases with atypical BL features or blastoid morphology without DH or TH cytogenetic findings, regardless of MYC status. They are phenotypically mature B-cell lymphomas. For cases with blastoid morphology, cyclin D1 and SOX-11 should be stained for to rule out blastoid variant of mantle cell lymphoma. Approximately 20–35% of HGBCL, NOS cases have a MYC rearrangement .
BLL-11q encompasses cases with morphologic, phenotypic, and gene expression resemblance to BL, but lacking MYC translocation and harbouring characteristic proximal 11q gains at 11q23.3 and distal 11q loss at 11q24-qter (Fig. 1b) [1, 3–5]. These 11q changes are likely to be the result in the upregulation of oncogenes PAFAH1B2, USP2, and CBL located in the gained regions of 11q23, and corresponding down-regulation of tumour suppressor candidate genes FLI1, ETS1, TBRG1, and EI24 located in the regions of 11q24-qter loss. Cases of BLL-11q commonly show some degree of cytomorphologic pleomorphism and tend to have a more complex karyotype than BL . The phenotype is that of GCB in nearly all cases [3, 4]. Besides the classic gains and losses within 11q, additional frequently identified abnormalities include del(6q) and trisomy 12 . A recent study has shown that BLL-11q lymphomas have mutational landscape distinct from BL , which indicates that BLL-11q is truly a distinct entity. BLL-11q cases occur over a wide age range but are more common in children and young adults. They are more frequently nodal than BL and tend to present as a single dominant mass or conglomerate mass [4, 10]. Patients tend to present with limited disease without involvement of bone marrow or central nervous system, and prognosis appears to be favourable, similar to classical BL .
LBL-IRF4 most commonly affects children and young adults [1, 5]. It mainly involves the Waldeyer’s ring and cervical lymph nodes and usually presents as low stage disease. Microscopically, the tumour cells are medium to large with finely clumped chromatin and small basophilic nucleoli. A starry sky pattern is usually absent, though proliferation rate is usually high by Ki-67 stain. These lymphomas may have a diffuse growth pattern, follicular growth pattern, or follicular/diffuse pattern. The tumour cells are positive for B-cell specific markers (CD20, CD79a, Pax-5), and characteristically show high levels of IRF4and BCL6. Over 50% of the cases are also positive for BCL2 and CD10. Despite the high levels of IRF4, these cases have a GCB signature by gene expression profiling. Most cases have a cytogenetically cryptic rearrangement of IRF4 with an Ig heavy chain locus. BCL6 alterations may be detected in some cases, but essentially all cases lack MYC and BCL2 rearrangement. Most cases have shown good response to chemotherapy [12, 13].
Diagnosis of these lymphomas
All the lymphomas described above except BL are uncommon and quite confusing to most general pathologists. They can be easily misdiagnosed in the absence of a proper work-up plan. Based on our practical experience, a step-by-step diagnostic algorithm has been designed, as shown in Figure 2. This diagnostic scheme is not recommended for the practice in BL endemic areas, where morphology and phenotype are likely to be adequate for the diagnosis of most BL cases. When a lymphoid malignancy is encountered with BL or BLL morphology, and mature B-cell phenotype is demonstrated by flow cytometry and/or immunochemical staining, the first check required is for MYC rearrangement. If MYC rearrangement is detected by FISH or other methods, BL will be the diagnosis in the presence of classic BL morphology and phenotype. If there is morphologic and/or phenotypic atypia, further testing for BCL2 and BCL6 rearrangement is necessary. In the presence of BCL2 and/or BCL6 rearrangement, the diagnosis will be HGBCL with DH or TH. Without the rearrangements of BCL2 and BCL6, the diagnosis will be HGBCL, NOS. Based on our experience in a children’s hospital in the USA, sporadic BL cases frequently show a certain degree of atypical morphologic features. And HGBCL with DH or TH is exceedingly rare in children [7, 8, 14]. Therefore, testing for DH/TH is usually not necessary for pediatric cases. These pediatric cases should be diagnosed as BL as long as MYC rearrangement is detected unless morphologic and phenotypic atypia is significant. The HGBCL, NOS category is not applicable for pediatric cases, considering the excellent prognosis of most pediatric HGBCL cases and their distinct molecular features [14–16]. In the pediatric population, MYC+ cases with significant morphologic and/or phenotypic atypia but no DT/TH should be diagnosed as DLBCL, NOS.
Although high Myc levels (in >80% of nuclei) are present in most cases of BL, there is much more variation in the HGBCLs with DH/ TH. Although most studies have concluded that Myc staining is not reliable enough to be used for the selection of cases for cytogenetic or molecular testing, some studies suggest using a cut-off of >30% or >70% Myc positivity for case selection [17, 18].
The rearrangements of MYC, BCL2, and BCL6 should be detected by a cytogenetic/molecular method such as FISH. The presence of only copy-number changes or somatic mutations, without an underlying rearrangement, is not enough to qualify a case for this category. So-called double-expresser DLBCLs show immunohistochemical overexpression of Myc and BCL2 protein and have a relatively poor prognosis . However, overexpression cannot be used as a surrogate marker for DH cytogenetic status, because most double-expressers are not DH lymphomas (although most DH lymphomas are also double-expressers).
If MYC rearrangement is absent (MYC−), the newly proposed entity, BLL with 11q, should be considered and properly tested. The diagnosis of BLL-11q is based on the presence of characteristic gain/loss patterns of 11q, together with BL/BLL morphology, GCB phenotype, and lack of MYC rearrangement. The characteristic 11q aberration is key to making the diagnosis, but its presence alone is neither specific nor diagnostic since it may also be present in MYC+ BL or DLBCL [10, 20]. The most sensitive method for detecting this characteristic cytogenetic finding is DNA microarray. The 11q aberration can be visualized by chromosomal analysis. However, chromosomal analysis relies on tumour cell viability and metaphase morphology, and the finding may not be characteristic for this aberration if the resolution is low. Another potential diagnostic strategy is FISH for chromosome 11 abnormalities. Commercially available FISH probes for chromosome 11 regions may be used to detect gains within 11q and 11q terminal loss. As a result of the variation of gain/loss spots among the cases, depending on the probes used, FISH alone may miss some cases. If MYC rearrangement is absent and there is no typical 11q aberration, the diagnosis should be HGBCL, NOS for adult patients. For pediatric patients, these lymphomas often have a Burkitt or intermediate molecular gene expression profile and show an excellent prognosis. These cases should not be classified as HGBCL, NOS. For the pediatric cases with morphology and phenotype close to BL and relatively simple karyotype, the diagnosis should be BL; otherwise the diagnosis should be DLBCL, NOS. If the case shows diffuse and levels of of IRF4 and BCL6, especially in Waldeyer’s ring and cervical regions, LBL-IRF4 with diffuse growth pattern should be considered. The diagnosis of LBL-IRF4 should be confirmed by FISH analysis for IRF4 rearrangement .
With the advances in cytogenetic/molecular studies of lymphomas with BL or BLL morphology, new classification of these lymphomas has been proposed. Accurate diagnosis of these lymphomas needs the combination of cytogenetic/molecular testing results, morphology and phenotype. More specific or targeted treatment for these lymphomas may be on the horizon, and hence accurately diagnosing them is clinically important.
Weijie Li MD, PhD Department of Pathology and Laboratory Medicine,
Children’s Mercy Hospital, University of Missouri-Kansas City School of
Medicine, Kansas City, MO 64108, USA
by Dr J. Filipe, Dr D. V. Pereira and Dr S. André
Granular cell tumours of the breast are rare, mostly benign neoplasms derived from Schwann cells. Clinical and radiological features may be worrisome, raising the possibility of malignancy. The only method for diagnosis is histopathological evaluation of the lesion, mandatory for the appropriate management of patients.
Granular cell tumours (GCTs) were first described in the tongue in 1926 by the Russian pathologist Abrikossoff, who suggested a myofibroblastic origin . He also reported the first case of breast GCT in 1932 . The 5th edition of the World Health Organization classification of breast tumors considers GCTs as mostly benign neoplasms with neuroectodermal origin, derived from Schwann cells . Rare cases of granular cell malignant tumours are reported in the literature, associated with poor prognosis with lymph node and lung metastases .
Breast tumours represent 8% of all GCTs, which are more common in the head and neck, proximal extremities, gastrointestinal and respiratory tracts . Usually, breast GCTs are single, but as many as 18% can be multicentric . They arise more often in young adult African-American women, with earlier presentation in this population (mean age: 41|years as compared with 54|years in white-Americans) .
The majority of cases occur in the upper inner quadrant and it is suggested that GCTs grow through intracanalicular branches of the supraclavicular nerve, around Schwann cells .
Most cases are sporadic but breast GCTs have been reported in association with other conditions in Bannayan–Riley–Ruvalcaba syndrome, neurofibromatosis type|1 and Noonan syndrome (also known as LEOPARD syndrome) . There are also reports of breast GCT associated with mastectomy scars and simultaneous GCT and breast carcinoma .
Loss-of-function mutations in ATP6AP1 and ATP6AP2 genes, involved in endosomal pH regulation, are frequently found in GCTs, leading to the accumulation of intracytoplasmic granules [3, 6]. These mutations are found irrespective of anatomic localization and are present in both benign and malignant tumours. As loss-of-function mutations in ATP6AP1 and ATP6AP2 genes were not yet described in other tumours, as far as it is known, they seem to be pathognomonic of GCTs .
According to recommendations by EUSOMA (the European Society of Breast Cancer Specialists), in the preoperative phase, histopathology evaluation of core biopsy is the gold standard for GCT diagnosis , as clinical and radiological features of breast GCTs mimic breast carcinoma [3, 7].
In fact, GCTs present as irregular firm masses, typically superficial and mobile, but may rarely be adherent to the pectoralis fascia. They may cause skin thickening, retraction and nipple inversion . On mammography, ultrasound and magnetic resonance imaging they are poorly defined masses with spiked margins but no microcalcifications [3, 7]. Despite these features usually associated with carcinomas only 1–2% of breast GCT cases show histological malignant change . It is uncertain if malignant GCTs are malignant transformations from benign lesions or occur ‘de novo’ .
1. Macroscopic appearance
Grossly, GCTs are white or tan, firm and homogeneous, with regular or spiked margins, that can reach up to 5|cm (Fig.|1) .
2. Histopathologic features
Different sampling methods allow histological evaluation of GCTs: (1) needle-core biopsy (Fig.|2a); (2) vacuum-assisted breast biopsy (Fig.|2b); (3) surgical specimens, such as lumpectomies (Fig.|2c).
Histologically, breast GCTs are poorly defined tumours with infiltrative borders (Fig.|2c), composed of sheets, clusters or trabeculae of large, round and polygonal cells, separated by collagenous bands (Fig.|3a). The cells have indistinct borders and may have a syncytial pattern (Fig.|3b).
The hallmark feature is the presence of abundant eosinophilic cytoplasm with granular appearance (Fig.|3b). The nucleus is centrally located and it is usually round, small, hyperchromatic, rarely vesicular and with prominent nucleoli. Mitoses are usually absent. Perineural and perivascular invasion is frequently present. When GCTS are localized in the dermis, they may be associated with pseudoepitheliomatous hyperplasia. In small and superficial cutaneous biopsies, this sometimes can be confused with squamous cell carcinoma [3, 6].
The finely granular cytoplasm is derived from lysosome accumulation. Larger intracytoplasmic granules with clear haloes, named pustulo-ovoid bodies of Milian, are usually periodic acid–Schiff stain (PAS) positive and diastase resistant [3, 6].
The rare malignant GCTs morphologically vary from having a sarcomatous appearance to relatively bland features . The histological criteria suggestive of malignancy are: increased cellularity, spindling, high nuclear to cytoplasm ratio, marked pleomorphism, vesicular nuclei with prominent nucleoli, more than two mitoses per 2|mm2 and geographical necrosis . Larger tumours (>5|cm) and local recurrence are also features favouring malignancy .
The histological differential diagnosis of breast GCTs includes reactive histiocytic lesions, dermatofibroma, epithelial tumours such as apocrine neoplasms and invasive carcinomas, melanocytic lesions (nevi and melanoma), hibernoma and alveolar soft part sarcoma [3, 6].
Morphological and immunohistochemical characterization are crucial in the differential diagnosis.
GCTs are strong and diffusely immunoreactive for S100 protein (Fig.|4a), CD68, CD63 antigen and neuron-specific enolase (NSE). The cells are also positive for transcription factor SOX-10 (SOX-10) (Fig.|4b), calretinin and inhibin A, and show diffuse nuclear expression of transcription factor E3 (TFE3) and microphthalmiaassociated transcription factor (MTIF). The cytoplasmatic granules are PAS positive and diastase resistant. Usually, there is no expression of cytokeratins (Fig.|4c), glial fibrillary acidic protein (GFAP), melanoma antigen recognized by T-cells 1 (Melan-A), HMB45- reactive antigen (HMB45), estrogen and progesterone receptors and receptor tyrosine-protein kinase erbB-2 (ERBB2) [3, 6].
S100 protein and SOX-10 are markers of Schwann cells but they may also be expressed in melanocytic lesions and primary breast carcinomas. The absence of cytokeratin expression excludes an epithelial lesion and the absence of Melan-A and HMB45 expression renders less likely a melanocytic lesion.
NSE is also a marker of neuroectodermal cells and is not specific for GCTs.
Inflammatory cells such as histiocytes can express CD68, as well as CD63 antigen; the last may also be present in neural derived tissue.
Dermatofibroma is also a benign infiltrative lesion commonly located at the dermis or subcutis with an identifiable grenz zone. It has spindle cells with scant cytoplasm and elongated nuclei dispersed among collagen bundles. Unlike GCTs, the cells are negative for S100 protein, SOX-10 and NSE.
Hibernoma is a benign, richly-vascularized adipose neoplasm composed by large brown fat cells with eosinophilic or pale multivacuolated cytoplasm that is granular with central nucleus, admixed with white adipose tissue. The cells are also positive for S100 protein, but staining is more variable. Unlike GCT, hibernoma is platelet endothelial cell adhesion molecule (CD31) positive.
Alveolar soft part sarcoma is a rare tumour of the soft tissue. It has an alveolar architecture and it is composed of large epithelioid polygonal cells with granular eosinophilic cytoplasm and prominent nucleoli. It has strong nuclear expression of TFE3, and may be focally positive for S100 protein; however, in contrast to GCTs, the cytoplasm has crystalline material and the cells are positive for actin and desmin. Furthermore, it is characterized by an ASPSCR1-TFE3 fusion gene.
Fine-needle aspiration (FNA) is often inconclusive. Smears are hypercellular, with large polygonal cells with fragile membranes and abundant eosinophilic granular cytoplasm and small regular nuclei; no stromal or myoepithelial components are present (Fig.|5) [3, 8].
The cytoplasm features can frequently be interpreted as apocrine benign lesions, histiocytic lesions or even apocrine, lobular and secretory invasive carcinomas [3, 8]. In contrast to apocrine cells, the cells of GCTs are larger, with more granular cytoplasm and poorly defined borders. Some cases may also have prominent nuclear atypia. The use of cellblock and immunohistochemistry can be crucial.
Moreover, owing to the rarity of breast GCTs, pathologists lack experience in its cytological evaluation . This method is no longer used for diagnosis of breast GCTs [5, 8].
Treatment of GCTs relies on local excision [3, 5]. The best approach is lumpectomy (Fig.|2c) or even excision by vacuum-assisted breast biopsy in small lesions, a relatively safe and minimally invasive procedure (Fig.|2b) [3, 9]. Sentinel lymph node biopsy is not recommended but may be considered in the rare cases of malignant GCTs . Metastasis have been described in 50% of malignant GCTs .
The recurrence rate is very low, even when excised with positive margins.
There is no need for adjuvant therapy in the benign cases, but long-term follow-up is recommended .
GCTs of the breast are rare neoplasms with neuroectodermal origin. Despite the fact that they are mostly benign tumours, they can mimic malignancies owing to their clinical and radiological features. Histopathology evaluation is the gold standard for diagnosis. Morphological and immunophenotypical features are characteristic, however differential diagnoses must be kept in mind.
Multidisciplinary approach is essential in breast tumours, and close contact between clinicians, radiologists and pathologists is vital for the correct management of patients.
Juliana Filipe* MD, Daniela Vinha Pereira MD, Saudade
André MD Serviço de Anatomia Patológica, Instituto Português
de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal
by Dr Jody M.¦W. van den Ouweland and Dr Rob Janssen
Desmosine is a promising biomarker for estimating elastin degradation activity in chronic obstructive pulmonary disease patients and provides a means to test the beneficial effects of therapeutic interventions. LC-MS/MS has emerged as a goldstandard method for accurate and sensitive measurement of desmosine in various body fluids, including plasma, urine, bronchoalveolar lavage fluid and sputum.
Chronic obstructive pulmonary disease (COPD) is one of the major health problems in the world, and currently the third leading cause of death by disease in the USA. COPD is a progressive lung disease defined by persistent airflow limitation predisposing the patient to exacerbations and serious illness. Distinct COPDphenotypes can be identified such as chronic bronchitis and emphysema. The disease is characterized by a low-grade inflammation and involves the release of enzymes that have the capacity to degrade the pulmonary elastic fibre network. Diagnosis is based on a combination of clinical symptoms and abnormalities in lung function tests. Chest radiology and arterial blood gas analysis are often used to establish disease severity. Validated lab tests that can be used in the management of COPD, however, are lacking. The current standard for determining COPD progression is through assessment of the decline of forced expiratory volume in one second (FEV1). As the rate of elastic fibre degradation is accelerated in COPD, matrix elastin degradation products may be effective biomarkers for estimating disease activity and to study the effect of therapeutic interventions . Elastin degradation is not unique for COPD and is also accelerated in several other chronic pulmonary conditions, including COPD, cystic fibrosis and tobacco use.
Principle of elastin synthesis and degradation
Elastin is a unique protein providing elasticity and resilience to dynamic organs, such as lungs and arteries and is thereby a basic requirement for both respiration and circulation. Elastin is synthesized in various cells which secrete the soluble precursor, monomer tropoelastin, into the extracellular matrix, which is then cross-linked mainly through formation of two amino acids, desmosine and isodesmosine (Dl), which are derived from the condensation of four lysine residues of elastin molecules by lysyl-oxidase (Fig. 1). The DI pyridinium ring has three allysyl side chains and one unaltered lysyl side chain (Fig. 2). Cross-linking transforms the soluble tropoelastin to the insoluble cross-linked mature elastin fibre. DI, as a cross-linker of elastin, gives elasticity to the tissue (Fig. 1). DI occurs only in mature elastin and its presence in body fluids is an indicator of degradation of mature elastic fibres .
Desmosine as a biomarker of elastin degradation
DI is one of the oldest discovered biomarkers and was developed in the 1960s, but the first time it was correlated to lung elastin content was in the 1980s. As the concentrations of DI in body fluids are extremely low, their precise and specific measurements have been a challenge. Initially, DI measurements in biological samples, particularly urine, relied on immunological techniques such as radioimmunoassay or ELISA as well as on spectrophotometric methods, all of them with limited selectivity and sensitivity, and inconsistencies in measured concentrations. Progressively, these methods have been replaced by more selective and sensitive methods such as capillary electrophoresis laser-induced fluorescence or liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowing measurement of DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum . Moreover, LC-MS/MS has shown much better inter-method agreement than other assays.
LC-MS/MS measurement of desmosine in body fluids
It has shown to be possible with LC-MS/MS to accurately measure DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum. The assay procedure for measuring total DI is rather laborious comprising three major steps including acid hydrolysis, solid phase extraction (SPE) with drying/resuspension, and LC-MS/MS. In brief, it starts with adding an equal volume of concentrated hydrochloric acid to plasma, urine or other body fluid including isotopically-labelled desmosine-d4 as internal standard, followed by a 24-hour incubation at 110|°C to liberate DI covalently bound to DI-containing peptides. Next, cellulose SPE is performed to extract total DI from plasma or urine after which the extract is dried and resuspended. Chromatographic separation of both isomers is achieved on a C18 column by addition of an ion-pairing reagent to the mobile phase, followed by selected reaction monitoring by mass spectrometry.
What was not anticipated were the many hurdles in the developmental process, taking years before the assay was ready to be used for clinical research in our hospital. First, the harsh acidic conditions used in sample preparation resulted in corrosion of stainless steel needles in the SPE manifold and in the dry-down heating block with consequent loss of peak signals. Second, discontinuation of critical SPE material by the manufacturer led to a long-lasting search for suitable alternatives. Finally, a twofold difference in measured concentrations of DI was observed when compared to data obtained from literature that could be traced back to an error in designation of the DI standard concen-tration by the supplier. Since then, our LC-MS/MS assay appears robust with performance of over 3000 analyses in various specimens and clinical application areas. The assay has a broad measuring range of 0.14–210|μg/L for DI enabling measurement in various body fluids.
Clinical application areas
We started our quest for an intervention to decelerate elastic fibre degradation. We studied the effect of vitamin|D administration on DI levels in COPD patients but did not find a favourable effect. From vitamin|D, we became interested in vitamin|K and were the first to demonstrate an inverse correlation between vitamin|K status and plasma DI levels . We found this association in patients with COPD and idiopathic pulmonary fibrosis (IPF) as well as in subjects using vitamin|K antagonists as anticoagulant medication. We are currently planning intervention trials in COPD to evaluate whether vitamin|K supplementation reduces DI levels. Elastic degradation accelerates during ageing and is particularly pronounced in COPD and IPF. Reference values for DI increase during ageing and have been established for non-smokers and smokers without lung diseases as well as for patients with COPD and IPF. DI levels appeared to be equally increased in IPF as in COPD . In cystic fibrosis patients, plasma DI correlated with lung function, exacerbation frequency and disease progression, suggesting that measuring DI levels in body fluids by LC-MS/MS may be an effective strategy of monitoring disease progression in cystic fibrosis patients .
A large study in 1177 COPD patients investigated the association between plasma DI and emphysema severity/progression, coronary artery calcium score and mortality . It was found that in COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. The latter may be due to the heterogenicity of the study population including distinct COPD-phenotypes from chronic bronchitis to emphysema. Indeed, elastin is not only present in alveolar walls but also in airways and plasma DI does not therefore specifically reflect emphysema formation. This can well explain why plasma DI was not correlated with emphysema progression in this heterogenous COPD population. Accelerated elastin degradation could potentially contribute to both the pulmonary and extrapulmonary disease manifestations of COPD and may represent a mechanistic link between COPD and the increased risk of cardiovascular disease.
Plasma DI levels correlate with emphysema severity on CT scan in patients with the genetic disorder alpha-1 antitrypsin deficiency (AATD). These patients have insufficient or absent AAT to protect elastic fibres from degradation by proteases, in particular neutrophil elastase. Weekly administration of alpha-1 antitrypsin reduced plasma DI levels . Given that loss of lung parenchyma is irreversible, early initiation in subjects with AATD and elevated plasma DI levels may be an attractive strategy to prevent permanent lung function decline. A plausible reason why plasma DI was correlated with emphysema in AATD patients and not in a heterogenous group of COPD patients, is that AATD patients are a rather homogeneous group with a common predominant form of panlobular emphysema in the basal lung fields.
Finally, in a recent study in SARS-CoV-2 patients, we found impaired vitamin|K-dependent matrix-Gla-protein activation, as a measure of extrahepatic vitamin|K status, linked to accelerated elastic fibre degradation and premorbid vascular calcifications as measured by DI in plasma . We are currently planning intervention trials in COVID-19 patients to evaluate whether vitamin|K supplementation improves outcome of SARS-CoV-2 infections.
In conclusion, the detection and measurement of DI as a means to study elastin degradation has been used for almost 30|years; however, recent methodological advances by our group and others have aided DI detection, as the concentrations present in body fluids are extremely low.
Conflict of interest statement
JO and RJ are owners of Desmosine.com. RJ discloses application of a patent for vitamin|K status as a prognostic and therapeutic biomarker in COVID-19.
Jody M.W. van den Ouweland*1 PhD and Rob Janssen2 MD
1Department of laboratory Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands
2Department of Pulmonary Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands
TECHNICAL NOTES & APPLICATIONS FOR LABORATORY WORK
The main flaw of traditional cell culture methods is the use of two-dimensional (2D) monolayers of cells, which are not a good representation of in vivo tissue environments. Today, there are various options for growing cells in 3D, including round-bottom surfaces, hydrogels or scaffold-based methods. However, for 3D cell culture to be assimilated at a faster rate by laboratories, there is a need for tools that enable 3D cell culture to be performed using routine 2D cell culture workflows. E.g. Tasks such as media exchanges and imaging cells can be challenging when working with 3D cell culture because cells are neither attached to plastic or grown on a flat surface. Magnetic 3D cell culture is the solution to perform 3D cell culture as easily as in 2D.
2/ GREINER BIO-ONE MAGNETIC 3D BIOPRINTING TECHNOLOGY
The principle of magnetic 3D bioprinting relies on the magnetization of cells with NanoShuttle™- PL, a biocompatible nanoparticle assembly of gold nanoparticles, ironoxide, and poly-L-lysine (PLL). The NanoShuttle™-PL magnetizes cells by electrostatically attaching to the cellmembranes via PLL. The reproducible formation of a single spheroid per well in an F-bottom plate with cell-repellent surface is induced by the magnetic forces of one magnet below each well within 15 min (Fig. 1). Then, spheroids are generally formed within hours, depending on the cell type. These structurally and biologically representative 3D models formed in vitro are ready to… Download white paper to continue reading