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
micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants
by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza
Sepsis is a condition that can develop and become life threatening very quickly. The key to obtaining the best possible outcome is early diagnosis and quick treatment, which can be challenging when treating very low birth weight infants and limited volume blood samples. This article describes how micromotors, tiny particles that can propel themselves autonomously, can be adapted to achieve immunoassays for sepsis diagnosis in a very short time and with a very tiny sample volume.
Sepsis in general and particularly neonatal sepsis in the highly vulnerable population of very low birth weight infants (VLBWIs), is still a major cause of mortality and morbidity. Despite the significant advances in neonatal care and the increased understanding of the pathogenesis of the disease in recent years, the ability to intervene and modify the path of the disease has been only partially successful.
An early sepsis diagnosis and timely initiation of the therapy improves patient outcomes significantly. However, the diagnosis of sepsis is still one of the fundamental challenges in healthcare worldwide . Early diagnosis is especially challenging in neonates owing to the low specificity and high variability of the symptoms, the lack of ideal sepsis biomarkers and the absence of optimal diagnostic tests . Blood culture continues to be the gold standard method for diagnosing sepsis. However, its low sensitivity, the delay in obtaining results and the relatively large sample volume needed from VLBWIs, make it unsuitable for an early sepsis diagnostic. Likewise, the limitations in the available volume of blood in newborns can negatively affect the performance of the blood culture, because of the low rate of bacteremia in most cases.
As a result of the aspects cited above, new diagnostic tools are much desired by clinicians. These analytical methods should provide specificity, sensitivity, multiplexed analysis and fast results at the bedside. Moreover, they should also account for the limitations in blood volume availability in VLBWIs.
Micromotor-based immunoassays: a new clinical analysis tool
To help in solving these challenges, the use of catalytic self-propelled micromotors, adequately functionalized with relevant specific antibodies, is proposed as a new paradigm in the clinical assay scenario for the analysis of validated sepsis biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP). The pairing of the well-known features of immunoassays and the great potential of micromotors provides a synergistic combination to develop highly interesting point-of-care-testing (POCT) devices for sepsis diagnosis. A brief introduction to the nature of micromotors is described before further elaborating on this specific application.
Micromotors are microscopic-sized nanotechnological particles that have the ability to move autonomously, offering a plethora of new possibilities in clinical analysis and other fields . Nature has provided many examples of its own machinery, such as kinesins, dynein, myosin or motor proteins to drive flagella and cilia in bacteria, sperm cells, etc. However, until the arrival of nanotechnology, the miniaturization of macroscopic objects was nothing more than a science fiction fantasy.
One of the main challenges of such micromachinery involves the power supply required for their propulsion. Although this energy can be provided by an external field or stimuli (ultrasound, magnetic and electrical field, light source, pH, temperature), the majority of micromotors developed so far harvest energy from the surrounding environment and are known as fuel-driven micromotors . These devices are constructed with an inner layer that is composed of a catalytic material which triggers a chemical reaction upon interaction with another chemical substance present in the liquid environment (fuel). Among others, the most widely explored mechanism for creating movement is bubble propulsion. In this case, a favourable geometry is selected for the design of the micromotor, such as a tube with one closed end and one open end. Then, decomposition of the fuel micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants Sepsis Diagnosis by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza A potential new clinical analysis tool for sepsis diagnosis: September 2020 21 | (i.e. hydrogen peroxide) on the inner catalytic surface of the micromotor tube causes the formation of gaseous product (oxygen) that creates a stream of bubbles. The exiting of the stream of bubbles from the open end of the tube creates a strong thrust, propelling the micromotor in the opposite direction. Bubble-propelled micromotors can move with high speed of up to several millimetres per second .
However, micromotor construction can be customized to perform the desired application. In this case, a layer-by-layer architecture was easily electro-synthetized via a template-assisted method to provide the required functionalities . Constructed in three layers, (Fig. 1) the external layer must contain carboxy moieties that can be further functionalized with the desired antibody to selectively recognize and capture the target protein and be able to successfully carry out all the immunoassay steps. The middle magnetic layer allows the guidance and stoppedflow operations of the self-propelled micromotors, providing easy manipulation/collection of the micromotor. The inner layer consists of the catalyst, which allows the bubble formation creating propulsion of the micromotor. The nanomaterials used for their construction can be chosen/selected accordingly to the desired properties .
Application of micromotors to sepsis diagnosis
With the knowledge of the techniques described above, our research group decided to apply this amazing technology to help solve the problem of sepsis diagnosis. Micromotor-based immunoassays have been used for the determination of both CRP and PCT levels, which are biomarkers relevant to sepsis diagnosis. Different nanomaterials have been used to construct the micromotors and different detection techniques have been tried, which demonstrates the versatility and potential of this technology.
Micromotor immunoassay detection of CRP
In a first approach, electrochemical detection of CRP using carbon-based micromotors was designed . Self-propelled catalytic micromotors functionalized with anti-CRP specific antibodies were designed for capture and electrochemical detection of CRP using a sandwich format, and horseradish peroxidase (HRP)-labelled tracer. Micromotors with different carbon-based outer layer compositions were evaluated as active supports for antibody immobilization while maintaining propulsion efficiency. Among them, reduced graphene oxide (rGOx) resulted in the highest affinity and the best immunoassay performance. Platinum nanoparticles forming the inner layer catalysed the oxygen bubble generation by the decomposition of hydrogen peroxide on its surface.
Once the successful micromotors have been synthetized, the immunoassay can be developed. The number of rGOx micromotors, the immunoassay performance (antibody concentrations, non-specific adsorption incubation times), and propulsion conditions have to be studied in depth. In contrast with conventional immunosensors, where the analyte interacts with the usually immobilized specific antibody by diffusion or external stirring of the solution, self-propelled micromotors actively move around the sample to bind the analyte. Besides the expected efficient movement of the micromotors, the generated microbubble tails can enhance the fluid mixing, and consequently improve the efficiency of the biorecognition event. In this sense, the analysis time can be decreased significantly (<10|min) and extremely low sample volumes (7|μL) can be used, in which other stirring mechanisms are not effective enough.
These features are especially relevant when small volume samples are available, such as those from preterm babies with suspected sepsis.
Briefly, the CRP-micromotor immunoassay (MIm) procedure consists of the addition of 10|μL of micromotor suspension (around 2000 micromotors) modified with the capture antibody into a test tube. Then, the sample and detection antibody were added (10|μL total volume, of which 7|μL was the sample) to perform the sandwich immunocomplex in one step. In order to generate movement of the micromotors, the fuel was also added. This fuel mix contained surfactant to lower the interfacial tension as well as H2O2 as fuel, which allows the catalytic reaction responsible for the bubble formation in the inner layer of the micromotor. Under these conditions, the modified micromotors swim around the sample to find and capture the specific analyte for 5|min, boosting the biological interaction thanks to their autonomous movement in such a small volume. As a result of their magnetic characteristics (intermediate magnetic layer of nickel), the micromotors can be stopped and retained while the supernatant was removed. Once the immunoaffinity interactions were complete, electrochemical detection can be easily accomplished, re-suspending the micromotor–immunocomplexes in just 45|μL of hydroquinone solution (electrochemical mediator) and magnetically captured onto the surface of a portable and disposable screen-printed carbon electrode. Amperometric measurements were performed after the addition of hydrogen peroxide solution as enzymatic substrate.
Under these circumstances, the obtained analytical characteristics are highly competitive with other CRP immunoassays reported in the literature . A linear working range from 2 to 100|μg/mL, (r|=|0.992), limit of detection (LOD) and limit of quantification (LOQ) of 0.8|μg/mL and 2.0|μg/mL, respectively, were obtained. Inter-assay precision for two different concentration values was 8%, whereas inter-assay precision was lower than 15% (n|=|5 days) for micromotors produced in different batches.
In order to evaluate the applicability in neonate clinical samples, the propulsion of the micromotors was tested in serum and plasma media, to compare with their behaviour in buffer. Although a diminished speed was observed in serum and plasma, it did not affect their function and indeed propulsion was improved in plasma by adjusting the fuel concentration. Hence, serum certified reference material (SCRM) and unique plasma samples from neonates with suspected sepsis were analysed.
The achieved results revealed an excellent accuracy of our MIm (Er|=|1%). Moreover, the results obtained by our MIm in comparison with those reported by the Hospital laboratory using standard analytical methods (BRAHMS CRPus KRYPTOR) for preterm neonate plasma samples, did not show significant differences (P<0.05), allowing the analysis of clinically relevant samples.
Seeking to fulfil all the requirements for performing on-site/bedside assays, the micromotor-based immunoassay for CRP described above was fully integrated into a microfluidic platform . In this new approach, both main immunoassays steps, immunocomplex formation and detection steps, were performed in the same microfluidic system, in a fully automated way, and so dispensing with the need for human handling. This on-chip MIm approach blends the advantages of the micromotors with those of microfluidic technology, giving place to a new alternative for sepsis POCT development. Among other advantages, this CRP assay is fast (<10|min), easy-to-use, automated, requires low sample volume (7|μL) and presents an improvement of an analytical characteristic (LOD|=|0.54|μg/mL). Furthermore, the good results obtained after SCRM (Er<10%) and the analysis of neonate samples demonstrated its applicability in a real clinical environment.
MIm detection of PCT
As previously stated, the flexibility of the technology allowed changing the target analyte by simply modifying the specific antibody bound to the outer layer of the micromotors, and the detection technique using an adequate label. In this sense, the second approach deals with a micromotor-based fluorescence immunoassay for PCT determination in samples from neonates . This time, different polymeric outer layers were tested, with COOH-polypyrrol being the polymer producing a higher degree of antibody functionalization and improving the sensitivity of the assay. The operating mode is similar to the previously developed CRP-MIm. However, in this case, detection is carried out on a fluorescence microscope by directly positioning 1|μL of the previously formed micromotor–immunocomplexes onto a microscope slide to perform the fluorescence measurements at excitation and emission wavelengths of 490|nm and 520|nm, respectively. These fluorescence signals were recorded and analysed by the high-resolution camera and its software associated with the microscope. A wide working linear range between 0.50 and 150|ng/mL, and LOD and LOQ of 0.07 and 0.50|ng/mL were obtained, respectively. Precision, even for intra- or inter-assay was lower than 9%. The applicability to real clinical samples was evaluated by analysing plasma samples from very low birth weight neonates. The results obtained by our MIm did not show significant differences with the PCT levels declared by the Hospital (BRAHMS PCT) (P<0.05).
In summary, these new micromotor-based immunoassay approaches exhibited key advantages such as simplicity, rapidity, miniaturization, automatization potential and reliability of analysis, using extremely low sample volumes and covering the entire range of concentrations involved in the clinical sepsis diagnosis (Fig. 2). Therefore, MIms are presented as future tools for early diagnostics, which is essential for timely treatment and the adequate guidance of antibiotic therapy as well as in the development of POCT devices.
Alberto Escarpa*1,2 , Miguel Ángel López 1,2 , Águeda Molinero-Fernández 1
PhD, María Moreno-Guzmán 3 and Luis Arruza 4
1Department of Analytical Chemistry, Physical Chemistry and Chemical
Engineering, University of Alcalá, Alcalá de Henares, E-28871 Madrid, Spain
2Chemical Research Institute Andres M. del Rio, University of Alcalá,
E-28871 Madrid, Spain
3Department of Chemistry in Pharmaceutical Sciences, Analytical Chemistry,
Faculty of Pharmacy, Universidad Complutense de Madrid, E-28040 Madrid,
4Division of Neonatology, Instituto del Niño y del Adolescente, Hospital
Clinico San Carlos – IdISSC, E-28040 Madrid, Spain
GAIN INDEPENDENT COMPENSATION
A biomarker for prognosis of critically ill surgical patients after sepsis
by Dr R. Stephen Smith, Nicole R. Mercier, Dr Scott C. Brakenridge
The best-known function of glucagon-like peptide 1 (GLP-1) is to promote insulin secretion in a glucose-dependent manner. However, GLP-1 also has effects in other tissues, notable the brain and stomach. Interestingly, GLP-1 is also linked to the immune system, and levels of GLP-1 have been shown to rise rapidly in response to cytokines, such as interleukin 6. Moreover, it is known that critical illness, such as sepsis, causes a disruption of glucose homeostasis. This article discusses how elevated levels of GLP-1 after the onset of sepsis seem to be a strong and independent predictor for the development of chronic critical illness and death in the subsequent 6 months.
Glucagon-like peptide-1 (GLP-1; Fig. 1) is a gut derived incretin hormone that stimulates insulin secretion, suppresses glucagon secretion, inhibits gastric emptying and decreases appetite. Loss of glucose homeostasis is a frequent occurrence in critically ill patients [1–3]. Numerous studies have demonstrated the association between loss of glycemic control and poor outcomes in the critical care setting [4–6]. Hyperglycemia is associated with increased mortality, increased rates of infection and increased length of intensive care unit (ICU) use . However, glycemic control is variable from patient to patient despite implementation of standard glycemic control protocols  and the pathophysiologic mechanisms for this variability are not fully understood.
Loss of glycemic homeostasis is a well-known phenomenon following critical illness and is associated with poor outcomes after trauma or sepsis [7–9], and we have previously found that GLP-1 levels were abnormal in seriously injured patients . Levels of GLP-1 during periods of physiologic stress secondary to sepsis has not been extensively studied or documented. Therefore, in our recent study, summarized here, we sought to determine the influence of sepsis on circulating levels of GLP-1 . We hypothesized that abnormal GLP-1 levels would be predictive of poor outcomes.
This is an analysis of an ongoing prospective longitudinal, observational cohort study of critically ill trauma and surgical intensive care unit (SICU) patients with sepsis at an academic medical centre. Overall programme study design and protocols have been previously published . GLP-1 biomarker analysis was performed on a group of 157 consecutively enrolled patients between January|2015 and September|2016. Patients included in this study were those SICU patients that screened positive for sepsis and were placed on our unit-specific sepsis protocol. Sepsis screening was performed using the Modified Early Warning Signs-Sepsis Recognition System (MEWS-SRS) . All enrolled patients were managed using a standard protocol based on the Surviving Sepsis guidelines.
Inclusion criteria were: (1) age ≥18 years; (2) SICU admission; and (3) diagnosis of sepsis, severe sepsis or septic shock . Exclusion criteria included: (1) refractory shock (death <24|hours) or inability to achieve source control; (2) pre-sepsis life expectancy <3|months; (3) goals of care not consistent with aggressive management; (4) severe congestive heart failure (New York Heart Association class 4); (5) Child–Pugh class C liver disease or pre-liver transplant; (6) HIV with CD4+ count <200|cells/mm3; (7) chronic corticosteroids or immunosuppressive agent use; (8) pregnancy; (9) chemotherapy or radiotherapy within past 30|days; (10) severe traumatic brain injury; or (11) spinal cord injury resulting in permanent deficits. Baseline and inpatient clinical data collected prospectively during initial hospitalization included patient characteristics, sepsis severity, clinical and laboratory data, complications and patient disposition.
Post-sepsis patient courses were categorized as ‘early death’, chronic critical illness (CCI) or ‘rapid recovery’ (RAP). Patients with refractory sepsis and death within 24|hours were excluded. Early death was defined as survival for 24|hours, but death before day 14. CCI is defined as an ICU stay of greater than or equal to 14|days with evidence of persistent organ dysfunction (Fig. 2), determined using components of the Sequential Organ Failure Assessment (SOFA) score (cardiovascular SOFA ≥1, or score in any other organ system ≥2) . Inpatient outcomes included in-hospital mortality, hospital and ICU length of stay, ventilator days, organ dysfunction and development of CCI. Six-month outcomes included performance status and mortality, which were determined by follow-up measured by the Zubrod scale.
All subjects enrolled underwent peripheral blood sampling at 12|hours, 1, 4, 7, and 14|days after sepsis onset, and weekly thereafter while hospitalized. These samples were processed and stored for subsequent programme a priori and ad hoc biomarker analysis. Analysis included GLP-1 (Luminex; MilliporeSigma) and interleukin-6 (IL-6) (ELISA; MilliporeSigma). We used IL-6 as a well-established measure of the host innate inflammatory response. We have previously demonstrated that IL-6 is a predictive biomarker for CCI after sepsis .
Data are presented as either frequency and percentage, or mean and standard deviation, or median and 25th/75th percentiles. Student’s t-test, ANOVA and Kruskal–Wallis tests were used for comparison of continuous variables as appropriate. Chi-squared test and Fisher’s exact test were used for comparison of categorical variables. Measured biomarkers were compared using non-parametric rank tests to determine significant differences between groups at each time point. Two multivariate logistic regression models were constructed to determine if GLP-1 was an independent predictor at 24|hours of the development of CCI, and at day 14 of death or severe disability (Zubrod score 4–5) at 6|months after sepsis onset. The models were designated a priori to model GLP-1 for these outcomes while controlling for both IL-6 (systemic inflammation) and peak glucose level (glucose dysregulation). Adjusted odds ratios with 95% confidence intervals (95% CI) and the area under the receiver operating characteristics curve values (AUC) and Hosmer–Lemeshow goodness-of-fit test were used. Statistical analyses were performed with SAS (v.9.4, SAS Institute).
Demographics, clinical course and outcomes
Over an 18-month period, 157 consecutively enrolled critically ill septic patients underwent circulating GLP-1 biomarker analyses. Overall, this cohort represents an older group of patients, with a significant comorbidity burden, a high incidence of early infectionassociated organ dysfunction (severe sepsis/septic shock, 64%) and severe physiologic derangement at 24|hours after sepsis onset. Approximately one-third of patients had pre-existing diabetes mellitus requiring either hyperglycemic control medications. Most of the patients were admitted with an acute intra-abdominal infection. The severity of organ dysfunction in this septic group of patients was high and the incidence of multiple organ failure was 50%. We excluded patients with limited goals of care, refractory shock and death within 24|hours; therefore, in-hospital mortality was lower than many previously reported sepsis cohorts (8%).
Approximately 60% of critically ill septic patients demonstrated rapid recovery from organ dysfunction and were discharged from the ICU within 14|days. Four patients (2.5%) suffered an early death, defined as less than 14|days of sepsis onset. The remaining 55 patients (35%) initially survived the episode of sepsis, but developed CCI, as evidenced by persistent organ dysfunction and a prolonged ICU length of stay (≥14|days). There were no significant differences in hospital admission diagnosis or septic source between the groups of patients. Compared to RAP patients, patients that developed CCI were older, had significantly higher pressor requirements for septic shock, and a greater severity of organ failure. Patients that developed CCI had a higher incidence of comorbidities than RAP patients, but there was no difference in the comorbidity rate of diabetes mellitus between CCI and RAP groups. Compared to RAP patients, those that developed CCI had significantly worse clinical outcomes (ventilator days, ICU days and mortality). Approximately 85% of patients that developed CCI survived to acute hospital discharge, but 90% of these survivors were discharged to facilities associated with poor long-term outcomes (long-term acute care, skilled nursing facility, another hospital, hospice). Post-discharge follow-up showed a 6-month mortality for the CCI group of 40%. In comparison, 6-month mortality for the RAP group was only 5%.
GLP-1 biomarker analyses
Owing to similarity of clinical course, the small number (n|=|4) of early death patients were combined with the CCI group for biomarker analyses. Circulating IL-6 levels were significantly elevated in all septic patients out to 28|days after sepsis onset as compared to healthy matched controls. Additionally, IL-6 levels were significantly elevated in CCI patients as compared to the RAP group at all time points between 24|hours and 28|days after onset of sepsis.
GLP-1 levels were significantly elevated among CCI patients at all measured time points from sepsis onset to 21|days. Maximum daily blood glucose levels were significantly elevated in CCI patients as compared to the RAP group across all measured time points. Accordingly, GLP-1 and blood glucose levels were modestly, but significantly correlated (Spearman correlation|=|0.27, P<0.0001). Logistic regression analysis revealed that GLP-1 level at 24|hours after sepsis onset was an independent predictor of CCI. Additionally, GLP-1 level at day 14 was an independent predictor for death or severe disability at 6|months.
Role of GLP-1 in glucose metabolism
Gut hormones facilitate metabolism of glucose through stimulation of insulin secretion and various other mechanisms . In normal physiology, plasma levels of GLP-1 increase soon after oral intake, whereas plasma levels of GLP-1 are low during fasting. However, the levels, trajectory and role of GLP-1 in patients with critical illness remains unclear. In murine models, increases in GLP-1 levels correlate with increased insulin secretion and reduced blood glucose levels after a glycemic challenge . The GLP-1 receptor is expressed in pancreatic islet alpha and beta cells and in a variety of peripheral tissues including the central and peripheral nervous systems, heart, kidney, lung and the gastrointestinal tract. Activation of incretin receptors on the pancreatic beta cells causes a rapid increase in insulin secretion , and, during normal physiologic states, this response occurs in a glucose-dependent manner . GLP-1 inhibits glucagon secretion and promotes glucose metabolism through neural mechanisms that contribute to glucose regulation. Antagonism of GLP-1 decreases insulin secretion and causes an increase in glucose levels . Additionally, it has been shown that administration of exogenous GLP-1 stimulates insulin secretion and causes glucagon suppression that results in decreased blood glucose levels .
GLP-1 receptors are abundant in many extra-pancreatic tissues, including the gastric, hepatic, cardiac and neural tissues. GLP-1 is an enterogastrone – meaning that it decreases gastric emptying in a dose dependent manner . GLP-1 also exhibits cardiovascular effects through increased inotropicity and chronotropicity as well as increasing blood pressure . GLP-1 receptors are also present in hepatocytes and appear to affect both glycogen and glucose metabolism . GLP-1 receptors are also found in the brain and nerves. Administration of GLP-1 into the cerebrum and ventricles of animals produces delayed gastric motility and gastric acid secretion .
Critical illness and loss of glucose homeostasis
It has been previously established that illness results in loss of glucose homeostasis. The hyperglycemia that occurs after trauma and serious illness impacts a number of physiologic systems including immune function, wound healing and other areas of metabolism. Acutely and critically ill patients with hyperglycemia have poorer outcomes [27–30]. The mechanisms responsible for glucose dysregulation in the ICU are complex, but is it seems apparent that levels of GLP-1 play an important role in glucose homeostasis. Deane et al. demonstrated that the exogenous administration of GLP-1 analogues in critically ill patients alters glucose metabolism [31–33]. Our evaluation of septic patients confirms these previous observations. Both GLP-1 levels and glucose levels were consistently and persistently elevated in patients that develop CCI. GLP-1 and blood glucose levels showed significant correlation in this group. The most straightforward explanation for this finding is that elevated GLP-1 levels were in response to high blood glucose. This is the well-documented function of the incretin system. GLP-1 receptors present on pancreatic islet cells increase insulin secretion. However, extra-pancreatic functions of GLP-1 appear to be involved in the response to sepsis. Our regression models demonstrated that an elevated GLP-1 level at 24|hours is an independent predictor for the development of CCI. Elevated GLP-1 levels 14|days after the onset of sepsis is a strong and independent predictor of death or severe disability in the subsequent 6 months. Surprisingly, this analysis showed that an elevated GLP-1 level is a better predictor of outcome than IL-6. Patients that developed CCI were older, more likely to require pressor support and had more medical comorbidities. However, the group that went on to CCI did not have a greater incidence of pre-sepsis diabetes. Again, this is suggestive of factors other than simple hyperglycemia causing a compensatory elevation of GLP-1 levels.
GLP-1 secretion has been demonstrated to rapidly increase in response to cytokines. For example, Ellingaard et al. showed that administration of IL-6 stimulates GLP-1 secretion from intestinal L cells and pancreatic alpha cells . This mechanism increased insulin secretion and improved glycemic control. This group concluded that IL-6 mediates crosstalk between insulin sensitive tissues, intestinal L cells and pancreatic islet cells to adapt to changes in insulin demand. LeBrun et al. noted that GLP-1 suppresses inflammation and promotes gut mucosal integrity. Furthermore, this group demonstrated that GLP-1 levels increased rapidly after the administration of lipopolysaccharide (LPS) in mice. This increase in GLP-1 was detected before measurable changes in cytokine levels or LPS. A similar response was noted after gut ischemia . Lebherz et al. have demonstrated the predictive value of GLP-1 levels in critically ill patients . This group measured GLP-1 levels in critically ill patients admitted to an ICU, patients with chronic kidney disease on hemodialysis and a control group without acute inflammation or kidney disease. Critically ill patients had a 6-fold increase in GLP-1 levels compared to the control group. Those requiring hemodialysis exhibited a fourfold greater GLP-1 level compared to controls. Lebherz concluded that both chronic and acute inflammatory states, including sepsis, increase circulating GLP-1 levels. This group further demonstrated in vivo that serum from critically ill patients had a strong potential for increasing GLP-1 secretion. Importantly, this group concluded that elevated GLP-1 levels were an independent predictor of mortality in critically ill and end-stage renal disease patients .
It is convenient to hypothesize that elevated GLP-1 levels are simply another compensatory biomarker of dysregulated glucose control after an acute, severe pro-inflammatory stressor. Another possibility is that GLP-1 elevation is representative of, or even contributory to, persistent low-grade inflammation and catabolism after sepsis. We have shown previously that a pathophysiologic syndrome of persistent inflammation, immunosuppression and catabolism is the mechanism driving the development of CCI, dismal long-term outcomes and mortality after sepsis [15, 16]. We have hypothesized that persistent kidney dysfunction perpetuates inflammation and immunosuppression through the local and systemic release of damage-associated molecular patterns, cytokines and the expansion of myeloid derived suppressor cells and also through metabolic reprograming (i.e. aerobic glycolysis) [37, 38]. After sepsis (or severe trauma) persistently elevated levels of GLP-1 may represent an underlying shift in metabolic programming towards aerobic glycolysis.
Elevated circulating GLP-1 levels within 24|hours of sepsis are a strong predictor of early death or the development of CCI. Elevated GLP-1 levels appear to be a better predictor of a poor outcome than IL-6. The predictive value of GLP-1 appears to be independent of other factors, such as hyperglycemia. Among early survivors, persistently elevated GLP-1 levels at day 14 are strongly predictive of death or severe functional disability at 6|months. Persistently elevated GLP-1 levels may be a marker of ongoing metabolic dysfunction and a non-resolving catabolic state. Future work should focus on elaborating the relationship of elevated GLP-1 to these underlying mechanisms.
R. Stephen Smith*1 MD FACS, Nicole R. Mercier2 MS, Scott C. Brakenridge1
1Department of Surgery, University of Florida, Gainesville, FL USA
2University of Arkansas for Medical Sciences, Little Rock, AR, USA