In Australia, burns are common, accounting for around 5500 hospital injuries each year. The proper management of burn pain is crucial to the rehabilitation process and in reducing the chance of long term psychological sequelae, such as depression and post-traumatic stress disorder. A wide array of therapeutic options is available to the clinician in managing burn pain in a hospital setting. These evidence-based options include opioids, non-opioid medications, anxiolytics, anaesthetics, as well as relaxation techniques and cognitive behavioural therapy. In managing chronic pain, therapeutic options vary between pharmacological and non pharmacological approaches used for acute pain. Consideration of these pain relief options can optimise the management of patients with burns and maximise their rehabilitation, leading to earlier hospital discharge.
Credit University of Western Australia
http://crowdresearch.uwa.edu.au/project/noisy-guts-project/
This review aims to summarise the current literature on employing exhaled breath volatile organic molecules (VOMs) as novel biomarkers for non-invasive testing in inflammatory bowel disease (IBD) patients. Inflammatory bowel disease is a multifactorial disease that significantly diminishes thequality of life of affected individuals. Currently, the tools employed in IBD diagnosis and monitoring are numerous, imprecise and invasive for patients. This has necessitated the need to develop new biomarkers that are accurate. The use of VOM breath testing is one such potential modality. This review discusses the efficacy of current IBD testing modalities and the principles of metabolic profiling. It evaluates the use of breath VOM profiling in IBD testing and postulates its implications for future practice. The VOM profiles of IBD patients are different to those of healthy individuals. VOM profiles also differ between IBD subcategories and correlate to disease severity. VOM profiling via the breath headspace is accurate, non-invasive and has the potential for point-of-care testing. VOM profiling offers an exciting avenue as a frontline diagnostic and monitoring tool for IBD patients and thus merits further research.
The primary goal of fasting prior to general anaesthesia is to reduce the risk of pulmonary aspiration, displacement of gastric contents into the lungs. As gastric volume, alongside patient age, current medications and type of surgery are associated with increased incidence of pulmonary aspiration. The preoperative fasting guidelines have been developed to reduce total fasting duration. Most recommend clear fluids up to two hours prior to surgery and solid food up to six hours prior to surgery. Reducing fasting time aims to minimize the negative metabolic effects of prolonged fasting, such as insulin resistance, catabolism, increased gastric acidity, discomfort, hypotension and dehydration. When combined with the negative effects associated with surgical trauma, many of these, particularly insulin resistance, have been associated with poor postoperative outcomes. Preoperative carbohydrate loading through the use of a glucose beverage has been examined as a method of reducing insulin resistance. There is a large amount of evidence suggesting it is a safe and effective preoperative tool. Patient compliance has been identified as a limitation of preoperative fasting guidelines, associated with a lack of understanding regarding their risk of pulmonary aspiration. Altering guidelines to include a default treatment program, consisting of carbohydrate treatment, minimum hydration requirements and enhancements in preoperative assessments to improve patient understanding, would likely improve patient outcomes.
Credit: Association of Community Cancer Centers
https://www.accc-cancer.org/acccbuzz/blog-post-template/accc-buzz/2017/10/05/the-ongoing-journey-improving-molecular-biomarker-testing-in-nsclc
Ongoing research increasingly reveals that metastatic colorectal carcinoma (mCRC) is a highly heterogeneous entity. Despite extension of the median survival of mCRC patients due to advances in therapeutic options available, further improvement and better rationalisation of resources could be achieved by more accurately predicting individual patient prognoses and responses to specific treatments. It is hence important to further our understanding of prognostic and predictive biomarkers in mCRC to enable accurate estimation of treatment benefit for individual patients and therefore guide patient selection. This information can also be used for improving patient stratification in future studies. The aim of this literature review is to highlight potential prognostic and predictive clinical, pathological and molecular biomarkers in mCRC. Broad categories include patient and tumour markers, protein markers and cell-free DNA, inflammatory markers and genetic markers.
The potential prognostic and predictive values of factors such as performance status, BRAF mutational status and neutrophil:lymphocyte ratio (NLR) >5 are supported by consistently strong evidence, but interpretation of the roles of other factors is difficult due to inconsistent findings between studies; however, many studies examine only small cohorts of patients, thereby limiting statistical power and variability in cut-off points may have contributed to different findings between trials. Although existing evidence may be used to select patient treatments and guide stratification in trials, future research with larger patient cohorts and clarification of appropriate cut-off values may prove helpful in elucidating the value of these biomarkers.
Credit: Psych Congress Network
https://www.psychcongress.com/article/new-clues-neurobiology-depression
Affective disorders, notably major depression and anxiety, are a significant cause of mortality and morbidity in society today, with the prevalence of depression estimated to be 10-16% in the general population and it is important to have effective treatments available for potentially life-threatening affective disorders. Yet, our understanding of the pathophysiology of depression and anxiety disorders has traditionally been limited due to the difficulty in investigating the brain in vivo. Thus, the molecular bases of these medication targets remain unclear. Recent advances in neuroscience have allowed us to gain a better understanding of the pharmacological basis of medical treatments for affective disorders. This new knowledge may pave the way for improved management of depression and anxiety. This review summarises some of the leading theories surrounding the neurobiology of depression and link them with both current and potential pharmacological treatments for depression.
Background: BRAF mutant metastatic melanoma treatment is most effective when it targets the changes induced within the mitogen-activated protein kinase (MAPK) cas-cade. However, due to the cancer’s heterogeneous nature, drug resistance predictably develops within 9-10 months, reducing treatment efficacy and producing poor patient outcomes. Understanding the mechanisms behind this acquired resistance is vital in de-vising optimal therapeutic regimens and ultimately improving the survival rate of this widespread disease.
Methods: This review examines the different resistance mechanisms that develop in BRAF mutant metastatic melanoma to prevent the durable efficacy of combined BRAF and MEK inhibitors as a treatment method. Subsequently, it evaluates possible changes that can be made to ensure therapy is made more effective in future disease management.
Results: Currently recognised resistance mechanisms include: alterations to BRAF (commonly through gene amplification), eIF4F complex mutations, changes activating the upstream regulator N-RAS or downstream effectors MEK1/2, and non-genomic al-terations. Together, these factors reactivate the MAPK cascade, despite dual MAPK in-hibition, and allow the tumour to continue to grow and metastasise unimpeded by inter-vention.
Conclusion: The ease at which contemporary treatment is being made redundant high-lights the requirement for further research into the underlying molecular aberrations, and from this, the development of new, more effective therapies into the future.
Introduction
Despite representing only 4% of all possible skin cancers, cutaneous melanoma has shown to be the most aggressive type, contributing to approximately 80% of all skin cancer related deaths (65,000 per year) [1]. It is the most common form of cancer diag-nosed in Australians aged 15-29 years, and its incidence in Caucasian populations has been increasing faster than any other malignancy over the last 30 years [2]. Five-year survival is poor for disseminated disease, from 15% to 60% in patients with either dis-tant or local metastases respectively [1,2]. Until 2011, treatment for patients with meta-static melanoma was largely ineffective, with chemotherapy having no effect on either median progression-free or overall survival [3,4]. However, an improved understanding of the underlying molecular aberrations in melanoma tumour cells led to the development of new targeted therapies, which have fortunately shown a significant clinical effect [2,5].
The genetic analysis of melanomas has revealed the clinical importance of BRAF, a pro-tein kinase that plays an essential role in activating the mitogen-actived protein kinase (MAPK)/extracellular signal related kinase (ERK)-signalling pathway [6]. Approximate-ly 40-50% of all cutaneous melanomas harbour activating BRAF mutations, although this aberration is rare in mucosal melanomas, and non-existent in the uveal form [7]. The mutation most commonly occurs in valine at codon 600, causing an increase in enzyme activity, which leads to angiogenesis, unchecked replication of cells and an ability to me-tastasise — all essential factors in tumour growth and spread [8,9]. As such, it is a vital target for drugs in reducing melanoma progression (and thus morbidity and mortality) when the diagnosis is made too late for simple excision. However, whilst treatments tar-geting a single aspect in the MAPK pathway were previously employed as a first-line defence, resistance to these therapies nearly invariably developed within 5-7 months of commencement [2,9-11].
Combined MAPK inhibition using both selective BRAF and allosteric MEK inhibitors as a method of circumventing these resistance mechanisms was subsequently consid-ered, and in 2014 met the approval of the Australian Therapeutic Goods Administration for disease treatment [3,10,12]. Initial clinical trials merging the BRAF inhibitor dabraf-enib, with MEK inhibitor trametinib, demonstrated an increased durability against drug resistance, thereby improving response rate, and enhancing progression-free and overall survival compared to single agent BRAF inhibition [7]. Following therapy initiation, 50% of patients experienced disease progression after 9-10 months, and long-term clini-cal outcomes were again impeded by the development of resistance [13,14].
Understanding of these resistance mechanisms is currently improving, however com-plete comprehension is vital if a significant reduction in melanoma mortality rates is to be achieved [5,15]. This review thus aims to provide readers with a thorough understand-ing of the resistance mechanisms that develop to the combined therapy of dabraf-enib/trametinib, and what improvements can be made to make treatment more effective in the future.
Discussion
Mechanism of action of dabrafenib/trametinib combined therapy in BRAF mu-tant metastatic melanoma
Normal MAPK function
The MAPK pathway (Figure 1) is a highly-conserved signalling cascade, essential for various cellular functions such as proliferation, differentiation and migration of cells [16-18]. Its activation is a complex process to ensure pathway regulation, and is often initiat-ed via RAS (a guanosine triposphate (GTP)-hydrolysing enzyme) binding to a GTP molecule, and leading to phosphorylation and activation of the RAF kinases [2]. These in turn phosphorylate the MEK1/2 kinases, which consequently enable ERK 1 and 2 ac-tivation [6,19,20]. ERK then proceeds to phosphorylate a plethora of cytoplasmic and nuclear substrates, which subsequently mediate the pathway’s pleiotropic effects. These include: cell cycle protein expression, proapoptotic and antiapoptotic regulation and function, and nuclear transcription factor activation [6,19-21].
Figure 1. The effect deregulation of the MAPK signalling pathway (left) and PI3K/AKT/mTOR pathway (right) has on melanoma tumourigenesis, and the various sites of intervention that occur with dabrafenib/trametinib dual therapy [21].BRAF mutation in melanomas
The RAF protein has three isoforms: ARAF, BRAF and CRAF [6,20,21]. As dis-cussed earlier, BRAF mutations occur at the highest frequency within most cutaneous melanomas (commonly as a V600E point mutation) and create a constitutively active BRAF molecule [23]. This subsequently leads to increased MEK and ERK function, which not only completely deregulates the cell cycle, but also increases transcription and inhibits apoptosis, thus inducing the cancer phenotype [23].
Mechanism of action of dabrafenib and trametinib
Dabrafenib is a highly potent, reversible adenosine triphosphate (ATP) competitive in-hibitor that selectively inhibits the kinase domain of mutant BRAF (Figure 1) [5,20,21]. This leads to reduced proliferation through subsequent reductions in phosphorylated ERK and thus, increased expression of apoptotic proteins and G1-phase cell cycle arrest [21]. Alternatively, trametinib is an allosteric inhibitor of MEK 1 and 2, that selectively binds and stabilises the closed, inactive conformation of the MEK enzymes, thereby re-ducing phospho-ERK concentrations and the downstream effects it has on cell prolifera-tion, growth and senescence (Figure 1) [18,24,25]. Ultimately, the combination of these two inhibitors significantly improved progression-free survival and response duration for metastatic melanoma patients when compared to monotherapy [12,14].
Mechanisms behind combined therapy resistance
The mechanisms involved in MAPK inhibitor resistance still largely require further in-vestigation. However, given the nature of combination therapy and the fact it was devel-oped to address many of the mechanisms causing acquired resistance in single target treatment, the culpable genetic aberrations are not as diverse as those seen in monothera-py [10,13,26].
There are currently several categories of mutations consistently identified as the source of MEK/ERK signalling reactivation: BRAF gene amplification, MEK1/2 mutation, and NRAS alteration [10,18,27]. There have also been a number of melanoma phenotypes recognised as possessing innate resistance, the mechanics of which are still under heavy investigation and only briefly covered within this report.
Resistance mechanism of BRAF gene amplification
BRAF copy number gains were most common, affecting 36% of all melanomas treated with combined therapy [10]. Notably, the extent of this amplification was significantly higher than seen in monotherapy resistance. Not only is this reflective of the upsurge in MAPK signalling required to overcome dual treatment, it is also indicative of increased tumour reliance on this resistance mechanism due to inhibition of other avenues [10,11,14]. Ultimately, gene amplification leads to augmented BRAF kinase concentra-tion within the cell, drastically elevating MAPK signalling by creating an excess of acti-vated MEK [8,14]. This has two vital consequences:
1. An increase in the basal level of phosho-ERK and thus increased activation of nuclear transcription factors, anti-apoptotic and cell cycle regulation proteins to cause proliferation, metastasis, and apoptosis resistance in melanoma cells [21,24].
2. An increase in the IC50 (the concentration of drug required to inhibit a biological process by half) of both trametinib and dabrafenib for inhibition of ERK phos-phorylation [6].
The latter point is explained by the mechanism of action of trametinib [14]. The drug has a significantly lower affinity for activated, phosphorylated MEK than it does for inactive MEK [10,14]. The presence of BRAF amplification and the resulting MEK hyperactiva-tion induces an excess of phosphorylated MEK, with little remaining in the drug’s fa-voured inactive conformation [10,24,25]. Overcoming this decreased affinity and ade-quately inhibiting MEK hence requires higher concentrations of trametinib [14]. Fur-thermore, because of the increased BRAF kinase concentration, therapeutic levels of dabrafenib cannot compete, and thus have an insufficient effect in inhibition [10].
Resistance mechanism of MEK1/2 mutation
Further implicated in resistance development were de novo mutations in MEK1/2, with an incidence of 26% in treatment-insensitive tumours [10,13]. The majority of MEK mu-tations employ a similar mechanism of action, with the alterations tending to occur within, or proximal to, a negative regulatory region of MEK; helix A/C sub structure [9,10,13,17]. The helix sits against the area of the kinase that binds both ATP and allos-teric inhibitors (such as trametinib). Whilst the mutations are usually located too far from the ligand to directly interact with it, they are close enough to alter the ATP binding site in a way that allosterically increases the intrinsic kinase activity of MEK [14,27]. As a result, ERK levels can be up to 20-fold higher compared to wild type MEK, increasing proliferation and apoptotic inhibition and ultimately creating an environment conducive to tumour growth [10,14]. Furthermore, over-expression of MEK causes a greater than ten-fold decrease in sensitivity to trametinib (due to mechanisms explained above), thereby increasing the concentrations required for MAPK inhibition and abrogating the effects of dabrafenib (which acts immediately upstream), and thus conferring acquired resistance to the combination therapy [10,13,27].
Coexisting NRAS mutation development
The emergence of coexisting BRAF and NRAS de novo mutations are also a possible cause for dual therapy resistance [28]. The NRAS gene encodes for the protein N-Ras, whose primary function involves regulating cell division. Its relatively high mutation rate means it contributes to the development of 15-20% of all non-uveal melanomas, and is subsequently the second most common oncogenic stimulus for cutaneous metastatic melanomas [7,28]. In around 80% of cases, these genetic aberrations involve a gain-of-function point mutation occurring at codon 61 of the NRAS gene, with the remaining alterations either affecting codons 12 or 13 [28]. This leads to a subsequent hyperactiva-tion of the RAS-RAF-MAPK and P13KT-AKT cascades, thereby increasing pro-survival protein expression, cellular proliferation, and cell cycle dysregulation [29]. The resulting synergistic effect of having two gain-of-function mutations within both NRAS and BRAF means a tumourigenic environment that supports metastasis quickly devel-ops, and the efficacy of trametinib/dabrafenib therapy is limited by the need to increase their required levels above what is therapeutically appropriate [28].
eIF4F eukaryotic translation initiation complex hyperactivation
Finally, it is important to consider downstream pathways and their influence on dual therapy drug resistance. MAPK and PI3K/AKT/mTOR signalling converge to influence the eIF4F eukaryotic translation initiation complex, a molecule nexus consisting of the eIF4G scaffolding protein, the eIF4E cap-binding protein and the eIF4A ribonucleic acid (RNA) helicase [30,31]. Its normal function involves modulating specific mitochondrial RNA translation to produce a plethora of proteins that potently regulate cell growth, pro-liferation, migration, and survival [31]. As such, excessive stimulation of the complex can alter the proteome, and ultimately give rise to the phenotypic heterogeneity of cells essential for drug resistance development, as well as tumour progression and metastasis [31,32]. There are three main mechanisms that have been identified in producing this augmented state, the first of which involves MAPK signalling reactivation through mechanisms described earlier [30]. Persistent phosphorylation of 4EBP1 (a protein that normally inhibits eIF4E binding) to permit increased translation initiation can also insti-gate hyperactivity, as can increased degradation of eIF4G through raised levels of pro-apoptotic BMF [30]. Ultimately, it is unsurprising that the enhanced formation and acti-vation of the eIF4F complex has been associated with dual therapy resistance in BRAF mutant metastatic melanoma, due to its propensity to produce the intratumoural hetero-geneity that helps enable drug resistance development [30-32].
Non-genomic resistance mechanisms
However, clinically acquired resistance to MAPK inhibition therapies cannot be fully explained through acquired genomic mechanisms, given that up to 10-20% of BRAF mutant metastatic melanoma patients never achieve a meaningful treatment response [7,27,33]. Rather, divergent transcriptional profiles exist between drug responsive cell lines and those which are intrinsically resistant, indicating that certain transcription fac-tors are innately present which can modulate melanoma response to MAPK inhibitors [7,29,33].
Differing tumour cell phenotypes and MITF
The significance of the microphthalmia-associated transcription factor (MITF) and its expression levels in treatment outcomes was one such identified transcription factor [34-36]. MITF plays a key role in the survival and differentiation of melanocytes by regulat-ing the expression of a variety of crucial melanogenic genes [35]. Whilst the majority of drug sensitive cells show high levels of MITF, both its expression and function were notably reduced within resistance lines. These cells instead tended to display elevated Nuclear Factor-κB (NF-κB) transcriptional activity, which in itself promotes melanoma progression and metastasis through pro-survival signalling [35,36]. The synergistic ef-fect of these phenotype factors creates a global transcriptional state that induces intrinsic indifference to intervention throughout all three levels of the RAF/MEK/ERK cascade. This was evidenced by the fact that, subsequent to dabrafenib/trametinib therapy, pro-gression-free survival of MITF-low/NF-κB-high melanomas was significantly shorter than the MITF-high/NF-κB-low group (median 5.0 months versus 14.5 months respec-tively) [35,36]. It should be noted however, that although this transcriptional state is cer-tainly associated with innate resistance, it can also be induced through MAPK hyperacti-vation, NF-κB induction and MITF dysregulation, thus becoming a mechanism of ac-quired resistance [36]. Ultimately, this transcriptional class distinction between BRAF mutant metastatic melanomas will aid future efforts in predicting treatment outcome and subsequently developing new therapeutic approaches for those patients unresponsive to RAF/MEK inhibition.
Further transcriptional alterations
There are a plethora of other transcriptional alterations that develop in treatment resistant tumours, frequently as the result of differential methylation of tumour cell-intrinsic cyto-sine-phosphate-guanine sites [7,28,37]. However, only the most recurrent molecular ab-errations will be discussed within this article: c-MET up expression, infra-physiologic LEF1 down expression, and YAPI signature enrichment [7,28]. Of the three, up expres-sion of c-MET not only remains the most consistently altered gene throughout treatment resistant melanomas, its degree of expression also greatly predicts patient outcomes via the mediation of MAPK-redundant survival signalling [37]. It is a receptor tyrosine ki-nase that reacts with its hepatocyte growth factor (HGF) ligand and stimulates an array of signalling pathways, ranging from proliferation to migration and invasion through activation of RAS and PI3K [37]. Evidently, its resultant hyperactivation of these path-ways ensures the level of BRAF and MEK inhibitors required to adequately control such a situation are too high to be within safe administration limits, thereby ensuring that melanoma cells carrying this mutation never respond to dual therapy [7,28].
Recurrent β-catenin-LEF1 down regulation has also shown to promote dual therapy in-sensitivity as its normally pro-apoptotic induction to MAPK inhibition is subsequently decreased [7]. Whilst this feature is essential for survival of metastatic cells, primary be-nign melanoma cells do not depend on this signalling cascade for survival. YAP1, a pro-survival factor that alters cell function through post transitional regulation, was also no-ticed to be harboured in increased quantities within MAPK inhibitor resistant tumours [7]. Given the history of known interactions between these two pathways in other bio-logical contexts, simultaneous deregulation of both β-catenin-LEF1 and YAP1 signal-ling is common, thereby resulting in an increased apoptotic threshold within melanomic cells, and thus reduced sensitivity to dual MAPK inhibition [7,28]. Given that the high-lighted transcriptional mutations are only a few of many in inducing therapy resistance, it is evident that current genomic diversity is severely limiting the long-term efficacy of dual medication.
Potential mechanisms to overcome acquired resistance in dabrafenib/trametinib combined therapy
Further MAPK pathway inhibition
MAPK-independent mechanisms of resistance were not conferred at a higher frequency in combined therapy compared to single-agent BRAF inhibitors [4,10,13]. This insinu-ates that BRAF mutant metastatic melanomas remain highly dependent on MEK/ERK signalling for tumour growth and survival, highlighting a potential avenue to increase combined treatment durability in the future and thus improve patient outcomes [10,14]. To elaborate, if other aspects of the pathway can be targeted along with BRAF and MEK inhibition, and thus the potency of MAPK inhibition further increased, it may help cir-cumvent the acquired resistance mechanisms which otherwise increase the concentration of activated MEK to levels dabrafenib/trametinib can no longer inhibit [10,12,13]. Ex-ample therapies include those targeting ERK through inhibition [4,26,27]. A preclinical study investigating this phenomenon found that BRAF/MEK/ERK inhibitor combina-tions not only delayed the emergence of acquired resistance, but they could also be used to overcome it in desensitised BRAF mutant tumours [27]. Whilst the exact reason for this is not yet clear, it is hypothesised that the ATP-competitive ERK inhibitors are less sensitive to altered conformation dynamics of activated ERK in the context of upstream oncogene amplification, and thus remain effective in inhibiting its downstream tumour-igenic effects on the cell [27]. Evidently, further refined investigation is needed into the area before more conclusive implications can be drawn, but current results allude to a hopeful future for a disease with such a poor survival rate.
Dual pathway inhibition
Furthermore, new studies have recently been released examining dual pathway inhibition [29,33]. The PI3K/AKT/mTOR pathway is an important cascade involved in signalling cellular growth, metabolism, and translation initiation (Figure 1). Along with MAPK, it is one of the most commonly altered signalling pathways in solid malignancy [33]. In melanoma cells, PI3K/AKT/mTOR has been shown to interact extensively with the MAPK pathway and potentially lead to its activation via phosphoionsitide 3-kinase (PI3K)-RAS interaction [29]. Whilst alone, it is not sufficient to completely confer re-sistance to combined therapy, PI3K has shown to contribute to earlier resistance devel-opment by modulating tumour responses to MAPK inhibitors [26,28]. Current evidence suggests that PI3K/mTOR pathway inhibition via ATP, and to a lesser extent, non-ATP competitive inhibitors can have a modest impact on both primary melanoma tumours and metastasis, diminishing the growth and proliferation of cells [29]. Therefore, the efficacy of PI3K and MAPK is being trialled in the hope it will ultimately improve patient out-comes [26,27]. Dual PI3K and MEK inhibition, the most common combination under-going investigation, currently results in only modest success and a number of relatively frequent adverse effects that include diarrhoea, nausea, pyrexia, rash, and fatigue. How-ever, this does not discount the feasibility of this dual pathway approach, but merely highlights the requirement for further investigation to improve tolerability [29]. This in-volves discerning the most optimal dosing schedule, perhaps targeting other members of the P13K/AKT/mTOR pathway, or augmenting patients with predictive factors [29].
Furthermore, the eIF4F complex (discussed earlier) sits at the junction of multiple onco-genic pathways and plays an essential role in producing the intratumoural heterogeneity that ultimately assists drug resistance development [32]. As such, its combined inhibi-tion with the MAPK pathway may provide a formidable effect in improving treatment efficacy [30]. There is currently work being done to target all three components of the complex, including: blocking eIF4E-cap interaction, interfering with eIF4E-eIF4G inter-action, inhibiting eIF4A helicase activity, and suppressing eIF4E levels — all with vary-ing degrees of success [31,32]. For example, eIF4A inhibitions (such as silvestrol) have a more potent effect compared to eIF4E in reducing global protein synthesis and thus the capability of tumour cells in developing treatment resistance, largely because cellular translation requires persistently high eIF4A concentrations, but not eIF4E, to be main-tained [30,31]. Whilst silvestrol therefore appears an advantageous agent to combine with MAPK inhibitors, it too is vulnerable to drug resistance due to overexpression of ABCB1/P-glycoprotein, thereby hampering its use in in vivo studies [30]. Ultimately, the introduction of eIF4F complex and MAPK doublet inhibitors as a possible treatment avenue is only a recent occurrence, and significantly more research is required before any definite conclusion can be established [31]. However, it theoretically can provide a potent influence in the seemingly insurmountable barrier of resistance in metastatic mel-anoma treatment, and thus provides hope for future patient outcomes [30].
Incorporation of immunomodulation in treatment
The potential of immunotherapy in treatment of metastatic melanoma has also been rec-ognised. Metastatic melanoma patients commonly display tumour-mediated immune suppression, and in the past, treatment with immunomodulatory therapies such as inter-feron alpha and high dose interleukin-2 has shown positive results [38]. With greater understanding of the immune system and its interaction with tumour cells, further inter-ventional therapies have now been developed with varying degrees of efficacy. These include monoclonal antibodies that inhibit essential immune checkpoints, such as ipili-mumab (an anti-cytotoxic T-lymphocyte-associated protein 4 inhibitor), and pembroli-zumab (an anti-programmed cell death 1 inhibitor), as well as other methods involving adoptive cell transfer [38]. Whilst it is hoped that a combination between these im-munomodulatory therapies with MAPK inhibition will improve clinical outcomes in metastatic melanoma patients, the toxic effects of such combinations currently remain unpredictable [38]. This only indicates the need for further, careful study into the dosing and timing of these dual treatments, as their potent anti-tumour activity and synergistic properties have high potential for improving patient outcomes.
Conclusion
Melanoma remains one of three cancers with an increasing mortality rate, despite exten-sive clinical investigation and the recent introduction of various novel and specific drugs into its treatment regimen [12]. This is primarily because long-term efficacy of these pharmaceuticals has been limited by the emergence of resistance in targeted cancer cells [3,5,6]. Given that the presence of BRAF mutations in metastatic melanoma has been associated with reduced survival in the absence of specific treatment, it is essential that these mechanisms are overcome to increase drug durability and thus improve patient outcomes [11]. It has been found that dabrafenib/trametinib associated resistance was primarily the result of BRAF gene amplification, MEK1/2 changes, the development of co-existing NRAS mutations, and eIF4F complex hyperactivation, as well as non-genomic alterations [10,13,14]. Together, they abrogate the effects of both drugs and cause resistance within 9-10 months of treatment commencement [21]. Possible solu-tions to overcome this include further inhibition of kinases downstream of RAF and MEK in the MAPK cascade such as ERK, as well as targeted inhibition of the heavily associated PI3K/ATK/mTOR cascade or eIF4F complex [10,26,28]. Incorporating im-munomodulatory therapies into current regimes is also a major point of consideration [36]. Ultimately, full comprehension of the factors influencing combination therapy re-sistance is fundamental, for only with understanding can solutions be developed, and the currently pitiable patient outcomes for BRAF mutant metastatic melanoma be improved.
As medical students enter the workforce, combination therapies will likely be the fore-front of metastatic melanoma treatment and only the beginning of the trend towards mu-tation-specific cancer management. As such, is it essential to have not only a strong un-derstanding of the basic pathways affected by these cancers, but the clinical relevance the short-term efficacy of these drugs have for patients. This need to be informed is only further augmented by the increasingly high incidence of melanoma in Australia.
Limitations of review
The major limitation of this literature review relates to the contemporary nature of the topic. With primary journal and review articles assessing the efficacy of dabrafenib/ tra-metinib still being released, investigations into the resistance mechanisms behind these results, and methods to overcome them, have only recently commenced. Consequently, there were a restricted number of articles available to examine which may affect the va-lidity of conclusions within this review when further information is made available. Fur-thermore, the sample sizes within the available primary journal articles were small [10]. Whilst the overlap in implicated mutations and possible solutions suggests valid results, it is possible that further vital mechanisms were missed because of the small sample size. Finally, it should be noted that whilst this review only focuses on dabrafenib/trametinib dual therapy, in actuality there are three BRAF/MEK doublets that have shown clinical benefit (dabrafenib/trametinib, vemurafenib/cobimetinib, encorafenib/binimetinib) [39].
Conflict of interest
None declared.
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Introduction: Sickle cell disease (SCD) is a genetic disorder impacting the patient’s haemoglobin. This condition is accompanied by many dangerous phenotypes, which are the result of pathological haemoglobin polymerisation within the red blood cell (RBC). The primary aim of this piece is to review the accepted literature on the SCD pathophysiology and its pharmacological treatment option, hydroxyurea.
Summary: Hydroxyurea has multiple posited mechanisms of action but most importantly it is the only SCD treatment that targets the underlying pathology. It was found that hydroxyurea significantly decreases the frequency of hospitalisations, vaso-occlusive events and blood transfusions required. In summary, SCD is a complex hereditary disorder in which for a medical practitioner to effectively manage requires a comprehensive understanding of both normal haemoglobin physiology and its pathophysiology.
Introduction
Sickle cell disease (SCD) is a common monogenic autosomal recessive disorder of haemoglobin, occurring in approximately 1-2% of Europeans and Americans of African descent and has a prevalence of 4% or higher in West Africa [1]. The primary role of haemoglobin is the transport of oxygen from highly oxygenated areas, such as the lungs, to the comparatively poorly oxygenated tissues. However, in SCD, low oxygen conditions result in the polymerisation of the pathological haemoglobin which downstream leads to the SCD signs and symptoms, including vaso-occlusive events, chronic haemolytic anaemia, organ dysfunction, and increased infections.
To extensively investigate SCD, this review will first address normal RBC physiology, the SCD pathophysiological models, and finally a treatment option for the condition. Regarding SCD treatment, this review will examine the pharmacological use of hydroxyurea, an antineoplastic agent. Finally, this topic is relevant and of considerable importance as it frequently leads to patient hospitalisations, life-threatening characteristics, and significant global prevalence.
Materials and Methods
A comprehensive review of literature was conducted using the computerised search databases, PubMed and Ovid MEDLINE. The obtained articles were then filtered and case reports and articles written in languages other than English were excluded. The review was conducted during September of 2016, with the most recent literature used where appropriate.
Normal physiology
The primary function of the RBC is O2 delivery from the lungs to the body’s tissues, mainly to allow oxidative phosphorylation in the mitochondria [2]. Within the RBC it is the chromo-protein haemoglobin that allows for the loading and unloading of O2. Haemoglobin contains four globin chains, with a corresponding haem molecule for which each has the ability to reversibly bind oxygen [3]. As haemoglobin binds O2 to its haem groups, its affinity for oxygen increases due to the alteration of the haemoglobin molecular structure. However, haemoglobin has more functions, those being; CO2 transport to the lungs from tissues as carbaminohaemoglobin, buffering of H+ via the carbamoyl anhydrase reaction within the RBC, and nitric oxide (NO) metabolism [3,4].
There are two types of adult haemoglobin, those being the major haemoglobin HbA and minor haemoglobin HbA2, comprising of two α-globin and two β-globin chains and two α-globin and two δ-chains, respectively [3]. These differing globin chains make HbA2 a considerably poorer oxygen carrier. Each of the α-chains are made of 141 amino acids, and β-chains of 146 amino acids. Chromosome 16 contains the genes for the α-chain and chromosome 11 has those for the β-chain [3].
The other type of haemoglobin is foetal haemoglobin (HbF) and is clinically and therapeutically significant in SCD, as discussed below. Structurally, HbF comprises of two α-globin and two γ-globin chains. Studies have shown that after 6 months, HbF begins to disappear from infant RBCs, however, the signalling mechanism of this is not known [5,6].
Pathophysiology
SCD is defined by the presence of sickle haemoglobin (HbS) in the RBCs, which causes the distinctive sickle RBC shape. SCD is an inherited disease done so in an autosomal recessive fashion. The carriers for the disease are heterozygous for the mutation and are said to have the sickle cell trait [7]. Patients that are disease compound heterozygous or homozygous have SCD and will exhibit some level of symptoms. In the U.S., the common SCD genotypes include: sickle cell anaemia (HbSS), HbS/β° thalassaemia, and HbS/β+ thalassaemia [7]. There are, however, many more compound heterozygous sickle cell genotypes, though most are rare.
HbS occurs due to a single nucleotide β-globin gene mutation, causing the 6th amino acid to be changed from glutamic acid to valine [8]. The mutation results in the binding of β1 and β2 chains of two deoxygenated HbS tetramers. This crystallisation process continues within the RBC, growing until the flexibility and structure is disturbed. Disease severity is dependent upon the degree of HbS polymerisation, which relies on the degree of haemoglobin deoxygenation and the concentration of intracellular HbS. For HbS, this deoxygenation results in the exposure of the mutated valine residue on the molecule surface, this causes hydrophobic interactions with surrounding chains. The consequently formed polymers develop into bundles, causing RBC distortion into its distinguishing sickle appearance. This reduces flexibility which impairs the flow of sickled RBCs through narrow vasculature.
The signs and symptoms of SCD can be understood via the three mechanisms: vascular occlusion, haemolytic anaemia, and increased infection tendency (Figure 1).
Figure 1. Sickle cell disease pathophysiology. The pathophysiological effects of sickled haemoglobin (HbS) polymerisation can be seen, being; vaso-occlusion, ischemia, haemolysis and decreased nitric oxide (NO) bioavailability. HbS polymerisation develops following RBC deoxygenation which consequently gives the RBC the characteristic sickle shape. Vaso-occlusion is the effect of interactions between the rigid sickled RBCs and the endothelial surface, causing downstream necrosis, organ dysfunction, acute pain and oxidative reperfusion stress. Haemolysis results in the release of haemoglobin into the plasma which removes bioavailable NO.
Vaso-occlusion most frequently occurs in the bones – but can occur anywhere – which is the result of sickled cells blocking blood vessels. Venous blockage results due to the physiological requirement for haemoglobin deoxygenation at the tissues which then allows for the formation of the pathological sickled RBC shape. Subsequent systemic disturbances and pain can be felt downstream from the blockage, caused by ischemia, necrosis and organ dysfunction. Fat embolisms can occur secondary to a bone marrow infarct which could exacerbate the vascular occlusion issue, especially in the respiratory and neurological systems [9].
Initially, it was thought that only the less soluble deoxygenated polymerised HbS caused the sickled RBCs to be ridged and dense, resulting in vaso-occlusion [10,11]. However, the process is multifaceted and dynamic incorporating stimulatory interactions of the vascular endothelium to increase numerous adhesion molecules, such as integrin. This produces an inflammatory response, RBC and leukocyte adhesion to vessel walls, as well as, tissue damage predisposing the patient to vaso-occlusion [12-14]. Such vaso-occlusion causes interrupted blood flow, oxidative reperfusion stress, thrombus formation, stroke, and potentially severe ischemia [15-18]. The later mentioned endothelial dysfunction which occurs due to decreased NO levels causing vasoconstriction, also contributes to this dangerous vaso-occlusive state [19].
Haemolytic anaemia is another marked pathology in sickle-cell disease, which too is promoted by HbS polymerisation. It is accepted that such haemolysis results in fatigue, anaemia, and cholelithiasis, and now is also being linked to the advancement of progressive vasculopathy. As patients with SCD age, their vasculopathy risk increases, primarily distinguished by pulmonary and systemic hypertension, as well as vascular intimal and smooth muscle proliferative modifications [20-22]. Clinically, the severity of intravascular haemolysis in SCD patients is determined by measuring the serum levels of lactate dehydrogenase [23].
Epidemiological studies have proposed a positive correlation to both low levels of non-polymerised haemoglobin and significant intravascular haemolysis with increased rates of the SCD manifestations, some of which include; cholelithiasis, cutaneous leg ulceration, priapism, and pulmonary hypertension [24,25]. Furthermore two prospective cohort studies have reported a relationship between pulmonary hypertension and haemolytic anaemia severity [24,26].
One pathophysiological basis for pulmonary hypertension in SCD is through the effect of haemolysed cells on NO and arginase. NO regulates vasodilation via the stimulation of cGMP dependent protein kinases, it also decreases platelet aggregation, and inhibits the release of endothelin-1, the powerful vasoconstrictor [27,28]. RBC haemolysis causes haemoglobin and the arginase enzyme to enter plasma circulation. This free haemoglobin is a potent NO scavenger, hence acting to impede the vaso-protective qualities of NO. Additionally, arginase catabolises arginine which is required for NO synthesis, hence perpetuating the decreased NO bioavailability [29]. Downstream arginase metabolites further increases vascular proliferation, inflammatory stress, and overall endothelial dysfunction [30]. These homeostatic changes will produce pulmonary vascular endothelial remodelling and constriction, hence resulting in pulmonary hypertension.
Another danger to SCD patients is their increased susceptibility to many encapsulated bacterial infections and is a major cause of mortality and morbidity [31,32]. The primary factors allowing this predisposition to encapsulated bacteria are: post-infarct hypo- or asplenia, abnormal opsonin phagocytosis due to potential defects in the alternative complement pathway, and deficiencies of particular circulating antibodies [33]. Common SCD infections are from Haemophilus influenza, non-typhi Salmonella, and Streptococcus pneumoniae [31,34,35]. The polysaccharide capsule of these bacteria acts to prevent the binding of complement or inhibits complement communication with macrophage receptors [36].
Treatment
As previously described, the underlying mechanism in SCD pathology is the polymerising of deoxygenated HbS, all stemming from the single nucleotide mutation of the β-globin gene.
Numerous management and treatment options are described in medical literature such as RBC exchange transfusions, however, hydroxyurea – an antineoplastic agent – is the only approved pharmacological intervention that treats the underlying SCD pathology and will be discussed at length for the remainder of this paper [37]. Hydroxyurea was approved for therapeutic use in 1998 by the US Food and Drug Administration in SCD patients suffering from frequent painful crises [38]. Despite much evidence supporting the clinical efficacy for SCD hydroxyurea treatment, its HbF induction mechanism of action continues to be largely unknown [39].
Hydroxyurea is a short-acting cytotoxic ribonucleotide reductase inhibitor which acts to arrest S-phase cells by impairing their DNA replication [40]. It may therefore enhance HbF production indirectly, by killing rapidly proliferating late erythroid cells [41,42]. The recurrent pharmacological injury to the erythropoietic marrow from repeated drug administration results in enhanced erythropoiesis, this increases primitive erythroid precursor recruitment, consequently raising HbF levels [43]. Most of the beneficial effects of hydroxyurea are attributed to the HbF induction, however, clinical improvement has been observed before a marked increase in circulating HbF, which has led to the postulation of additional mechanisms of action, including decreased intercellular adhesion enhancing blood flow and increased NO bioavailability. Hydroxyurea metabolism is the cause of this increase in NO levels [41].
It has been known for some time that the primary effect of hydroxyurea, increased HbF levels, can ameliorate SCD [8]. This works by its reduction of RBC sickling because HbF replaces the mutated β-globin with a γ-globin chain which is non-pathological, hence decreasing the number of vaso-occlusive events and infarction. Even if an infarct occurs, the decreased circulating neutrophils from hydroxyurea administration may regulate the degree of tissue damage and pain felt [44]. There is also evidence that hydroxyurea generates NO and increased cGMP levels which causes the induction of foetal γ-globin mRNA and HbF protein [45].
Hydroxyurea has been shown to downregulate the expression of specific adhesion molecules on vascular endothelium [46]. This phenomenon is evidently independent of any of the SCD β-globin gene effects. Considering the importance that exposure to hypoxic capillary bed venules has on sickling, and consequently vaso-occlusive events, any reduction in endothelial-sickled RBC adhesion would have salutary effects.
Pregnancy is contraindicated with the use of hydroxyurea because it is known to have mutagenic, carcinogenic, and teratogenic effects in animals [47]. However, this relationship was not observed in 94 pregnancy outcomes of the human SCD patients that participated in hydroxyurea drug trials that despite precautions became pregnant mid-trial [47]. This suggests the foetal exposure to therapeutic hydroxyurea may not be teratogenic. Conversely, another study indicated abnormal sperm parameters in males taking hydroxyurea that possibly could have teratogenic effects or cause infertility [48]. It is important to note, however, that most individuals in this study had abnormal sperm prior to commencing hydroxyurea treatment, thus it was difficult to establish the precise contribution of hydroxyurea. Therefore, considerably more research and follow up time should be allocated to pregnant SCD subjects exposed to hydroxyurea to determine definitive results.
At present, there have only been 5 randomised control trials conducted regarding the efficacy of hydroxyurea treatment in patients with sickle cell anaemia. Of these, all except the Stroke With Transfusions Changing to Hydroxyurea (SWiTCH) trial indicated benefit for hydroxyurea treatment over the standard of care [49-53]. Study sizes range between 44 and 299 participants. The results from these studies described reduced vaso-occlusive crises, pulmonary pathologies, and blood transfusion frequencies in those receiving hydroxyurea treatment (Table 1) [49,50,52,53].
Table 1. SCD hydroxyurea randomised control trials. Abbreviations: HU = hydroxyurea, y = year, SCA = sickle cell anaemia, VOC = vaso-occlusive crisis, ACS = acute chest syndrome, Hx = history, mo = months, MTD = maximum tolerated dose.
The Belgian paediatric randomised control trial showed that hydroxyurea treatment resulted in statistically significant increases in levels of HbF and decreased both hospitalisations and vaso-occlusive events when compared to the placebo [49]. Wang and associates found that although changes in renal and splenic function were insignificant, there were statistically relevant decreases in vaso-occlusive events, hospitalisations, and the number of blood transfusions required [52]. A small Indian based study also reported that children treated with hydroxyurea experienced decreased numbers of vaso-occlusive events, hospitalisations, and blood transfusions required, despite the low doses of drug administered [50]. The findings from the Multicentre Study of Hydroxyurea, conducted by Charache et al [53], too demonstrated decreased incidents of vaso-occlusion, hospitalisations, and required transfusions compared to the placebo. However, in Charache et al [53], results were compromised by the substantial loss to follow up, with only 134 of the initial 299 participants completing the full 2-year trial.
At present no phase 3 randomised control trials are enrolling individuals with different genotypes to HbSS and HbS/β° thalassaemia [38]. However, two cohort studies from Italy and Greece have shown that hydroxyurea efficacy observed in HbSS individuals also extends to those with HbS/β+ thalassaemia [54,55]. However, this hydroxyurea recommendation is weak due to the limited sample population when likened to the size of HbSS data.
Conclusion
To conclude, this literature review has discussed the normal physiology of haemoglobin, pathogenesis of sickle cell disease as well as its course of treatment. It was found this haemoglobinopathy is responsible for vaso-occlusive crises, pulmonary pathologies, and increased susceptibility to infection. Nonetheless, hydroxyurea was reviewed as a potential treatment option. Hydroxyurea has multiple posited mechanisms of action but more importantly is the only SCD treatment that targets the underlying pathology. There is opportunity for future research into both the exact hydroxyurea mechanism of action in SCD patients and hydroxyurea efficacy and dangers when used to treat SCD patients expressing one of the rare genotypes. Thus, to summarise, SCD is a complex hereditary disorder in which for a medical practitioner to effectively manage requires a comprehensive understanding of both normal haemoglobin physiology and its pathophysiology.
Conflict of interest
None declared.
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Introduction: This article aims to provide a narrative review of the most recent primary literature on the pathways associated with the neuroprotective effects of remote ischaemic post-conditioning (RIPC) in stroke and discuss the prospect for application in the clinical setting.
Summary: This narrative review identified multiple pre-clinical in-vivo studies. These studies found that RIPC modulates a wide variety of pathways within the brain, including those associated with inflammation, reactive oxygen species (ROS) production, antioxidant regulation, and oedema development. Modulation of these pathways was associated with a significant reduction in the neuronal damage, a finding supported by measured reductions in cerebral infarct volume and apoptotic neuronal populations. Clinical research was limited; only one trial on RIPC in stroke was discovered. Whilst the results were positive the small sample size of the study does not make them definitive.
RIPC as an intervention for ischaemia reperfusion injury (IRI) in stroke has been found to be considerably effective in animal models, stimulating a wide variety of neuroprotective pathways. The limited clinical research has not yet been able to confirm RIPC efficacy in human stroke models but should be a catalyst for further research.
Introduction
Cerebrovascular disease constitutes the third greatest cause of mortality in the Australian population, with 23 people dying from a stroke every day [1]. Ischaemic stroke occurs when an artery, supplying a particular area of the brain, is blocked by an embolus, starving neuronal tissue of oxygen and resulting in permanent damage of the local area [2]. Treatment for ischaemic stroke usually involves thrombolysis which dissolves the embolus and restores blood supply to the ischaemic area [1]. Although reperfusion is a critical intervention, it is also associated with a spike in neuronal cell damage, in a phenomena known as ischaemia reperfusion injury (IRI) [2,3]. In recent years, considerable effort has gone into developing ways to limit the impact of IRI. One promising therapy is remote ischaemic conditioning (RIC) [4].
The principle of RIC is to expose the brain to a series of sub-lethal cycles of ischaemia and reperfusion through intermittent vascular occlusion of another distant and accessible organ such as the upper or lower limb [5]. Transient ischaemia performed in this manner is known to activate several endogenous neuroprotective pathways which reduce the damage associated with an IRI [5,6]. RIC can be performed before the onset of ischaemia (“pre-conditioning”), during the ischaemic event (“per-conditioning”), and after reperfusion (“post-conditioning”) [5]. By their very nature, both pre- and per-conditioning require pre-emptive action which is simply not practical in the health care setting. As such, this review focuses on remote ischaemic post-conditioning (RIPC) performed on the femoral artery in the time immediately following a stroke [7]. This review will explore and integrate the pre-clinical literature surrounding the neuroprotective pathways of RIPC and thus produce a narrative on how this novel intervention acts on a molecular level, and how it may translate to clinical medicine.
Ischaemia reperfusion injury
IRI is a multi-faceted process, in which reperfusion is associated with cellular death, rather than the restoration of normal function. This abnormal response is a consequence of the ischaemia induced dysregulation of cellular function [8]. One such example is the dysfunction of the Na+/K+ ATPase which triggers the accumulation of intracellular sodium. Sodium accumulation is an initiator for a series of events including calcium overload, excitotoxicity, acidosis, cellular swelling, and the initiation of apoptosis [4].
Another event which occurs during prolonged ischaemia is damage and dysregulation of the mitochondrial electron transport chain (ETC). Once a cell undergoes reperfusion, the ETC will again utilise oxygen to try to produce ATP. However, due to extensive damage, a high proportion of this oxygen will instead be metabolised to form reactive oxygen species (ROS) [9]. The excessive ROS production overwhelms the brain’s anti-oxidant system, resulting in lipid peroxidation, dysregulation of proteins, DNA damage, and alterations in transcription [8].
Ischaemia also has the effect of priming the endothelium for leukocyte recruitment through increasing adhesion molecules and the transcription of pro-inflammatory factors, such as NFκB. Upon reperfusion, these adaptations will stimulate the recruitment of neutrophils to the ischaemic area, where they will release ROS and further exacerbate oxidative damage and cell death [10].
Figure 1. Basic inflammatory pathways resulting in apoptosis and necrosis of neuronal tissue in IRI [2-8]. ETC, Electron transport chain; ROS, Reactive oxygen species; NFκB, Nuclear factor κB.Collectively, these effects of cellular dysfunction, ROS production and inflammation result in the death and destruction of neurons even after blood supply has been restored (Figure 1). Whilst little can be done about the tissue destroyed by the initial ischaemic event, a reduction in IRI may have the potential to prevent the death of otherwise salvageable neurons [2,3].
Discussion
HIF1a/TIM3 axis in the initiation of inflammation
Inflammation is a key contributor to the damage associated with IRI, with the recruitment of leukocytes responsible for the substantial production of ROS and cytokines [10]. The inflammatory response of cerebral IRI is thought to be mediated in part by Hypoxia Inducible Factor 1a (HIF1a), which is activated and up-regulated during and after cerebral ischaemia [11]. Zong et al [11] investigated the effects of RIPC on HIF1a, discovering that RIPC significantly reduced the expression of HIF1a when compared to the control group one day post reperfusion. These results correlated with a 10.3% reduction in cerebral infarct volume [11], suggesting that HIF1a is an important target in the neuro-protective effects of RIPC [11,12].
The mechanism in which HIF1a mediates its downstream effects is still a topic of some speculation [11,12]. Recent research by Koh et al [13] investigated how HIF1a interacts with another downstream signalling molecule, TIM3. It was found that in response to hypoxia, HIF1a binds to the TIM3 prompter region to increase its expression. Increased TIM3 up-regulates the inflammatory cytokines IL1b and CXCL1 (Figure 2) [13]. Blockage of TIM3 activity by monoclonal antibodies showed a significant reduction in the expression of both these cytokines as well as a substantial reduction in cerebral infarct volume. The findings of Zong et al [11] and Koh et al [13] suggest that RIPC exerts its neuro-protective effect via inhibition of HIF1a; subsequently resulting in down-regulation of TIM3 and reduction in cytokine and ROS production [10-13].
Figure 2. Activation of the HIF1α/TIM3 axis producing inflammatory cytokines and neutrophil recruitment [11-13]. HIF1α, Hypoxia inducible factor 1α; TIM3, T-cell immunoglobulin and mucin domain protein 3; IL1-β, Interleukin 1β; CXCL1, Chemokine CXC ligand 1; ROS, Reactive oxygen species.
PKCd, JAK2/STAT 3 and P38 MAPK apoptotic pathways
Apoptosis is a process of organised cell death, triggered by an extensive number of signalling cascades. One well known example involves PKCd, an intracellular enzyme, activated by cell stress to initiate apoptosis [14]. It has been extensively reported that RIPC exerts an anti-apoptotic effect via reduction of PKCd expression [3,5,14]. However, recent research has investigated several additional pathways which may be involved in the anti-apoptotic effects of RIPC. One such pathway is the JAK2/STAT3 axis which has been found to attenuate apoptosis in multiple models of IRI [15,16]. Cheng et al [17] recently investigated the potential effects of RIPC on JAK2/STAT3, finding that it was associated with a significant increase in both JAK2 activity and STAT3 protein expression. The increase in STAT3 expression results in the transcription of anti-apoptotic Bcl2 family proteins, which inhibit the formation of Bax channels and the initiation of apoptosis [15-17].
As well as its effect on anti-apoptotic signalling, Cheng et al [17] also demonstrated that RIPC was potentially involved in the JAK2/STAT3 mediated inhibition of NFkB. The inhibition of NFkB results in the reduction of pro-inflammatory cytokines such as IL1b and TNFa; subsequently reducing recruitment of neutrophils and other leukocytes to the brain [3,17]. The collective effects of RIPC on JAK2/STAT3 mediated reduction of inflammation and apoptosis, resulted in a 13.2% reduction in rodent cerebral infarct volume when compared to the IRI control group (Figure 3) [17].
Figure 3. RIPC mediated activation of the JAK2/STAT3 pathway causing inhibition of pro-apoptotic and pro-inflammatory pathways [15-17]. LRIPC, Limb remote ischaemic post-conditioning; JAK2, Janus kinase 2; STAT3, Signal transducer and activator of transcription 3; IL1β, Interleukin 1β; TNFα, Tissue necrosis factor α.Another recently investigated apoptotic pathway involves the p38MAPK enzyme, responsible for phosphorylation of downstream signalling molecules in response to cellular stress [18]. Li et al [19] investigated the effects of RIPC on p38MAPK on in vivo rodent models. They found that the RIPC intervention was associated with down-regulation of this enzyme as well as a reduction in IRI induced apoptotic neuron populations. Li et al [19] proposed that the attenuation of apoptosis occurred through the RIPC/p38 MAPK mediated reduction of the transcription factor ATF2. How this reduction in ATF2 results in an anti-apoptotic effect is, however, unclear. Other conflicting literature has found that it is the up-regulation of ATF2 which is associated with Bcl2 production, and hence a reduction in apoptosis [20,21]. Although the exact mechanism remains a topic of speculation, many studies do support this study’s conclusion that P38 MAPK down-regulation is associated with a reduction in apoptosis [18-21].
PI3k/Akt and eNOS uncoupling
Endothelial nitric oxide synthase (eNOS) is responsible for the physiological production of nitric oxide (NO), a key regulator of normal endothelial cell function. During IRI eNOS can become uncoupled and as a result transfer its electrons to an oxygen molecule to produce superoxide free radicals [22]. These superoxide radicals can cause direct cellular damage but can also combine with NO to produce peroxynitrite, a highly destructive reactive nitrogen species (RNS) which suppresses protein function in the same way as ROS [8,22].
The process of eNOS uncoupling is thought to result from both ROS mediated damage and the depletion of the eNOS substrate BH4 [22,23]. Chen et al [22] investigated the capacity of RIPC to attenuate these effects; finding that it was associated with both an increase in BH4 availability, as well as a reduction in ROS (through inhibiting transcription of NADPH oxidase and xanthine oxidase). The RIPC mediated inhibition of eNOS uncoupling resulted in a significant reduction in peroxynitrite levels, as well as a 15.1% reduction in cerebral infarct volume when compared to the control group [22].
RIPC has also been found to directly increase the synthesis of the eNOS enzyme, leading to higher levels of NO during reperfusion [22,24,25]. It is thought that this effect may be associated with the RIPC induced up-regulation of the PI3k enzyme, which triggers phosphorylation of the protein kinase B (Akt) transcription factor. Akt is an important anti-apoptotic signalling factor, which is also thought to upregulate eNOS transcription [24,25]. The increased production of eNOS increases the quantity of NO produced within the ischaemic brain; this promotes vasodilation and reduces inflammation and thrombosis [22,24]. Furthermore, the reduction in eNOS uncoupling increases the proportion of NO to superoxide radicals produced [22]. Hence, it appears that RIPC works synergistically to upregulate eNOS transcription whilst reducing uncoupling, resulting in an increase in NO and reduction in ROS and RNS [8,22-25].
Nrf-Antioxidant response element (ARE) pathway
Small amounts of ROS are produced in normal physiological processes, but cause no damage because they are rapidly neutralised by cellular antioxidants. The excessive ROS production in IRI overwhelms the antioxidant system and leads to extensive cellular damage [8,26]. Li et al [26] investigated the effects of RIPC on the Nrf2-ARE pathway. It was found that RIPC increased the activity of Nrf2 which subsequently increased ARE transcription [26]. ARE increases the production of the antioxidant molecules SOD, HO1, and NQO1 which, through a range of different cellular processes, neutralise oxygen free radicals into less reactive substrates (Figure 4) [27,28]. RIPC mediated neutralisation of ROS correlated with a significant reduction in MDA (a lipid peroxidation marker), as well as a 16.9% reduction in cerebral infarct volume compared to the control group [26].
ATP deficiency during the period of ischaemic stroke triggers dysfunction of the Na+/K+ ATPase, accumulation of intracellular sodium, and influx of water into the astrocytes. Fluid accumulation within the astrocyte produces cytotoxic oedema, an event which results in death and destruction of nearby tissue [3,4]. Recent research has shown that this influx of water during IRI is likely mediated by the aquaporin 4 (AQP4) channels [29]. Li et al [30] has found that RIPC can reduce the expression of AQP4 channels in the brain following reperfusion, thus reducing the influx of water into the astrocytes. Reduction in astrocyte swelling was found to attenuate cytotoxic oedema formation, resulting in a 15.0% reduction in rodent cerebral infarct volume compared to the control group [30].
Cytotoxic oedema usually occurs in the earlier stages of IRI, when the blood-brain barrier (BBB) is still intact [3,4]. Later in disease progression, there is an increased production of ROS through the mechanisms which have previously been discussed [22,24,26,31]. ROS have been found to mediate BBB damage through oxidative damage, tight junction modification, and activation of matrix metalloproteinases (MMP) [32]. As a result of these events, BBB permeability increases, leading to influx of fluid into the neuronal extracellular space in an event called vasogenic oedema [31,32]. Li et al [30] demonstrated that RIPC may reduce BBB permeability and hence reduce vasogenic oedema: a finding supported by another study investigating RIPC in carotid stenosis [33]. RIPC most likely reduces BBB permeability through a variety of pathways targeting inflammation, ROS and antioxidant production that may all impact the integrity of the vascular endothelium or initiate endothelial dysfunction [29-33].
Integrating the pathways
RIPC is an expanding field with more pathways being discovered each year. As this occurs, it is important to know how each pathway relates to another (Figure 5).
Figure 5. Interconnection of the neuroprotective pathways proposed for RIPC, ultimately leading to reductions in apoptosis, necrosis and neuronal cell death, post reperfusion [11-33]. LRIPC, Limb remote ischaemic post-conditioning; SOD, Superoxide dismutase; HO1, Heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase; Nrf2, Nuclear factor erythroid 2-related factor 2; ARE, Antioxidant response element; JAK2, Janus kinase 2; STAT3, Signal transducer and activator of transcription 3; IL1β, Interleukin 1β; TNFα, Tissue necrosis factor α; HIF 1α, Hypoxia inducible factor 1α; TIM3, T-cell immunoglobulin and mucin domain protein 3; CXCL1, Chemokine CXC ligand 1; ROS, Reactive oxygen species; ETC, Electron transport chain; NFκB, Nuclear factor κB; GTPCH, GTP cyclohydrolase I; BH4, tetrahydrobiopterin; AQP4, aquaporin 4; P38MAPK, p38 mitogen-activated protein kinase; PI3K, phosphoinositide 3 kinase; Akt, Protein kinase B; PKCδ, Protein kinase C δ; eNOS, Endothelial nitric oxide synthase.
RIPC in the clinical setting
The application of RIPC in animal cerebral ischaemia models has been demonstrated to be safe and efficacious, reducing cerebral infarct volume and apoptotic neuronal populations substantially (Table 1) [5,7,34].
Table 1. Results from pre-clinical trials.
Whilst the preclinical evidence for RIPC is convincing, there is still a large gap in the translation to clinical trials. In May of 2017, the results of the RECAST trial were published. This phase I pilot blinded placebo controlled trial involved 26 patients with ischaemic stroke, 13 of whom were given the RIPC intervention within 24 hours of ischaemic stroke. Whilst small, this pilot study found that RIPC was very well tolerated, safe, and feasible in the clinical setting. Furthermore, RIPC was found to provide significant improvements in neurological function when compared to the control group [35]. As the only study published of its kind, the evidence for RIPC in stroke is still tentative, however, a recent meta-analysis on the use of RIPC in acute coronary syndrome has provided some more firm results. The analysis of 13 clinical trials demonstrated that RIPC was effective in reducing infarct size, reperfusion injury, and improving patient outcomes post myocardial infarction [36]. These recent publications provide a convincing argument for the further clinical exploration of how RIPC can be utilised in patients who suffer from ischaemic stroke.
Current gaps
Confusion surrounding the molecular mechanism of RIPC primarily pertain to a gap in understanding how an episode of ischaemia in the lower limb can result in an increased expression of neuro-protective factors within the brain [6]. Current research, predominantly conducted on cardiac models, suggests that this effect is likely achieved through multiple neurological, humoral, and immune events. How these mechanisms relate to ischaemic stroke and how they result in such diverse effects is still unknown [6,37-39]. Further research must be conducted, particularly in cerebral ischaemia models, before links can be made regarding the initiation of the RIPC neuroprotective pathways.
Conclusion
IRI is a major contributor to the death and destruction of the neuronal tissue after ischaemic stroke. The RIPC intervention aims to attenuate this damage through stimulation of neuroprotective pathways within the brain. Although clinical trials remain limited, RIPC has shown substantial efficacy in the pre-clinical setting: reducing cerebral infarct volume and apoptotic neuron populations. Research should continue to be conducted regarding the pathways involved in RIPC, however, a shift must also take place in translating this pre-clinical knowledge into clinical trials. The limited clinical data is positive thus far, but more must be done to determine whether this intervention is appropriate for the clinical setting.
Conflict of interest
None declared.
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Introduction: Subchorionic haematoma (SCH) is the most common ultrasound abnormality found in women with symptoms of threatened miscarriage. It refers to a collection of blood between the chorionic membrane and the uterine wall. Depending on the time the haematoma is formed, it may appear as either hypoechoic or hyperechoic on the ultrasound. The cause of SCH may be related to poor placentation. Although SCH is common, the effects of SCH on pregnancy outcomes are unclear. The review aims to provide an overview of the effects of SCH on pregnancy outcomes and identify prognostic factors that may predict adverse pregnancy outcomes in women with SCH.
Methods: To identify the relevant literature, electronic databases (PubMed and EMBASE) were searched using the search terms: “subchorionic haematoma” and “subchorionic hemorrhage”. Exclusion criteria include multiple pregnancy, ectopic pregnancy, Breus mole, review articles, case reports, and studies that did not focus on the effects of SCH on pregnancy outcomes.
Results and conclusion: Women with SCH have an increased risk of placental abruption. Studies are conflicting on the risk SCH poses on pregnancy loss. There are only limited studies on other adverse pregnancy outcomes such as preterm delivery, small for gestational age, pre-eclampsia, and chorioamnionitis. Factors that may predict an increased likelihood of adverse pregnancy outcomes in SCH include: large haematoma size, fundal or retroplacental location, early gestational age of diagnosis (before 9 weeks), and severity of symptoms. Persistent SCH is rare but it carries a high risk of complications, including chorioamnionitis.
Introduction
Up to 25% of pregnant women experience symptoms of threatened miscarriage, namely first trimester per vaginum (PV) bleeding with or without uterine contractions [1]. The most common ultrasound abnormality in these women is a subchorionic haematoma (SCH) [2]. SCH is a collection of blood between the chorionic membrane and the uterine wall [2]. Typically, it appears as a crescenteric hypoechoic lesion around the gestational sac [2]. According to a 2014 retrospective cohort study from Turkey, the reported incidence of SCH in women with symptoms of threatened abortion is 18.2% [3]. In the general obstetric population, the incidence of SCH varies between 1.7% to 3.1% [4,5].
Pathophysiology
The exact pathophysiology of SCH is still unknown. Nevertheless, the underlying cause of SCH is believed to be poor placentation [3,6]. Poor placentation can impair angiogenesis and lead to the formation of weak vessels that tear easily [3,6]. In SCH, it is postulated that the marginal utero-placental veins tear and cause low pressure bleeding [7]. The blood then tracks around the gestational sac to form a cresenteric haematoma between the chorionic membrane and the uterine wall [7]. In contrast, the bleeding in placental abruption is usually high pressure bleeding from ruptured spiral arterioles [8]. Results from a recent Japanese study appear to support the theory that SCH is caused by poor placentation [9]. The study found that SCH is more common in women with risk factors for poor placentation such as multiparity and pregnancies conceived through in-vitro fertilisation, especially those using a frozen-thawed embryo transfer [9].
Clinical presentation
While some SCH are asymptomatic, most can present with first trimester PV bleeding with or without uterine contractions [5,10]. In most cases (up to 70%), PV bleeding of varying degree, ranging from spotting to heavy bleeds, can continue intermittently for 1 – 3 months after the diagnosis of SCH [11]. The symptoms usually resolve spontaneously during the second trimester [11]. However, a small minority of women (0.46% of all obstetric patients) can have a persistent SCH that remains symptomatic until delivery [11].
Diagnosis
SCH is diagnosed using ultrasound (Figure 1). The characteristic sonographic finding is a hypoechoic cresenteric lesion between the chorionic membrane and the uterine wall [2]. The haematoma may appear hyperechoic initially but with time, it becomes hypoechoic [2]. Possible differential diagnoses for this sonographic finding include chorioamniotic separation and twin gestational sac [2].
Figure 1. First trimester subchorionic haematoma.
Rationale and aims
Although SCH is very common in women with symptoms of threatened miscarriage, the effects of SCH on pregnancy outcomes are unclear. This review aims to provide an overview of the effects of SCH on pregnancy outcomes. It also aims to identify prognostic factors that may predict adverse pregnancy outcomes in women with SCH.
Methods
To identify the relevant literature, electronic databases (PubMed and EMBASE) were searched using the search terms: subchorionic haematoma and subchorionic hemorrhage. The search was limited to English-language human studies published between January 1981 and June 2016. A total of 192 studies were identified from the database search and an additional 14 studies were identified from manual review of bibliographies. After removing the duplicates, studies were excluded for reasons such as multiple pregnancy (n = 2), ectopic pregnancy (n = 2), Breus mole (n = 11), review articles (n = 2), case reports (n = 4) and different study focus (n = 82) (Figure 2). For this review, 28 studies discussing the effects of SCH on pregnancy outcomes were included.
Figure 2. PRISMA study flow diagram.
Effects on pregnancy outcomes
Adverse pregnancy outcomes that may be associated with SCH include early and late pregnancy loss, placental abruption, preterm delivery (PTD), preterm premature rupture of membrane (pPROM), small for gestational age (SGA) and chorioamnionitis. The pathophysiological mechanisms behind how SCH might contribute to these adverse pregnancy outcomes remains unclear. It is thought that SCH may cause pregnancy loss through either a direct mechanical effect or an indirect inflammatory response [12]. As for placental abruption, studies suggest that the underlying cause of SCH – poor placentation – also predisposes the patients to placental abruption [5,13].
Early and late pregnancy loss
In several studies, SCH is associated with an increased risk of miscarriage (pregnancy loss before 20 weeks) and stillbirth (pregnancy loss after 20 weeks). In a recent study, Sukur et al. [3] reported that the miscarriage rate was significantly higher in women with SCH compared to those without (29.5% versus 12.6%). A similar finding was obtained by Kurjak et al. [14]. According to Ozkaya et al. [15], the risk of miscarriage was six times higher in women with SCH (OR = 6.29, 95% CI 1.43 – 37.7). In a separate study, the risk of miscarriage remained significantly higher in cases of SCH even after PV bleeding has stopped [16]. Stillbirths are also more common in women with SCH compared to women without SCH [13]. Ball et al. [13] found that the risk of stillbirth was significant when compared to women both with and without PV bleeding. From a meta-analysis performed in 2011, SCH doubled the risk of miscarriage and stillbirth (OR = 2.18, 95% CI 1.29 – 3.68 and OR = 2.09, 95% CI 1.20 – 3.67, respectively) [6]. Based on the meta-analysis, for every 11 women with SCH, there was one additional miscarriage [6].
However, some studies had contrasting results. Two small studies (n = 22, n = 62) of SCH did not find that SCH increases the miscarriage rate [17,18]. In one of these studies, there were no miscarriages in all 22 cases of SCH studied [17]. Tower and Regan [19] studied the effect of SCH in a population with recurrent miscarriages. They found that SCH did not increase the miscarriage rate for these patients [19]. Based on a 2003 prospective study, women with SCH also did not have a significantly higher risk of stillbirth [5]. In a large retrospective study, the risk of stillbirth in women with SCH was not significant after adjusting for ethnicities, PV bleeding, chronic hypertension, pregestational diabetes, and smoking [4].
The mixed results from different studies suggest that the effect of SCH on pregnancy loss is complicated. Not all cases of SCH have an equal risk of miscarriage and stillbirth. The likelihood of pregnancy loss in SCH may depend on several prognostic factors, such as the size and location of haematoma.
Placental abruption
Unlike the risk of pregnancy loss, the risk of placental abruption in women with SCH is well established. When SCH is present, the risk of placental abruption increases from 0.6% to 3.6% (aOR = 2.6, 95% CI 1.8 – 3.7) [4]. This finding is echoed by several other studies [5,13,20]. From a meta-analysis that pooled together the results of four different studies, SCH increased the risk of placental abruption by more than fivefold (OR = 5.70, 95% CI 3.91 – 8.33) [6]. The number needed to harm was only 34 [6]. Given that studies have consistently reported that women with SCH have a higher risk of placental abruption, SCH most likely has a true effect on the risk of placental abruption.
Preterm delivery (PTD)
Studies also found that women with SCH have a significantly higher risk of PTD [4,5,20,21]. In 2003, Nagy et al. [5] reported that SCH doubled the risk of PTD (RR = 2.3, 95% CI 1.6 – 3.2). In their study, 43% of these PTD cases occurred before 34 weeks and 10% occurred before 28 weeks [5]. This result was corroborated by a large retrospective cohort study from 2010, which included more than 1000 cases of SCH [4]. More recently, Palatnik and Grobman [20] carried out a multivariable regression analysis and showed that SCH increased the risk of PTD independent of mid-trimester cervical length. There were several studies that did not find a correlation between SCH and preterm delivery [3,13,14,18,19]. However, these studies were smaller in size, with only one of the studies having more than 100 cases of SCH [13]. Nevertheless, more studies are required to confirm the risk of PTD in women with SCH.
Chorioamnionitis
Chorioamnionitis is a rare but severe complication in pregnancy that can cause life-threatening neonatal sepsis. Currently, the risk of chorioamnionitis in women with SCH is still unknown. There was only one study that extensively investigated the risk of chorioamnionitis in SCH [11]. In that study, Seki et al. [11] reported that chorioamnionitis was particularly common amongst women with persistent SCH. Six out of 22 women (27.3%) with persistent SCH had chorioamnionitis [11]. Half of these women had a miscarriage, while the other half delivered preterm [11]. In the study, persistent SCH was defined as a haematoma with clinical symptoms that lasted until delivery [11]. Recently, a study found that women with SCH had significantly different vaginal swab culture results [22]. Women with SCH had significantly higher prevalence of coagulase-negative Staphylococcus and Gardnerella vaginalis and lower prevalence of Lactobacillus on vaginal swabs [22]. The culture result is suggestive, though not diagnostic of bacterial vaginosis, a condition that has been associated with chorioamnionitis, pPROM, and PTD [22]. However, in that study, the swabs were only collected in the second trimester, which was temporally distant from the time SCH was diagnosed [22]. Hence, a direct cause and effect relationship could not be confirmed through the study [22]. The risk of chorioamnionitis in women with SCH warrants further investigation.
Small for gestational age (SGA) and pre-eclampsia
Given that SCH may be associated with poor placentation, it is important to also consider other adverse pregnancy outcomes typically associated with poor placentation such as SGA and pre-eclampsia. SCH was associated with a significantly higher risk of SGA in two studies [5,15] and and pre-eclampsia in one study [5]. However, the majority of the studies did not support a significant relationship between SCH and SGA [3,4,12,13,19,20]. SCH was also not associated with pre-eclampsia in multiple studies [4,12,19,20]. More importantly, in the two largest controlled studies on SCH (n = 512 and n = 1,081), SCH did not increase the risk of SGA or pre-eclampsia [4,20].
Preterm premature rupture of membrane (pPROM)
Limited studies recorded the incidence of pPROM in women with SCH. In a study by Palatnik and Grobman [20], pPROM was significantly more common in women with SCH compared to women without SCH (6.4% versus 4.0%). However, this finding was not reciprocated in two other studies [4,12].
Other adverse pregnancy outcomes
Interestingly, Nagy et al. [5] noted that women with SCH had significantly higher rates of an abnormally adherent placenta that required manual removal (13.9% versus 4.9%). Previously, two uncontrolled studies also noted that manual placenta removal was required in 7% and 11.1% of women with SCH [10, 23]. More controlled studies are needed to provide information about the risk.
Prognostic predictors
The likelihood of adverse pregnancy outcomes in SCH may depend on several prognostic factors. Differences in the size, location, and persistency of SCH, as well as, the gestational age of diagnosis and the severity of symptoms can all change how SCH affects pregnancy [24]. Examining these factors in closer detail can help clinicians clarify the risk of SCH.
Size of haematoma
The risk of adverse outcomes may be increased with larger haematoma size. In the original case series on SCH, Mantoni and Pederson [25] noted that SCH > 50mL occurring after 16 weeks gestation increased the risk of spontaneous abortion and PTD but SCH < 35 mL had a good prognosis. In one study, all women had SCH < 16 mL and none of them had a miscarriage [17]. In contrast, another study found that 81% of the pregnancies with SCH > 60 mL did not continue to term [21]. The rate of miscarriage appeared to be vastly different depending on the size of the SCH. Ozkaya et al. [15] used a receiver operating characteristic curve analysis (ROC) and determined that haematoma > 32 mL was 81% sensitive and 80% specific for predicting the risk of miscarriage. The size of haematoma was also shown to be an important factor for miscarriage in several other studies [23,26-29]. However, many studies did not observe an association between size and pregnancy loss [1,13,14,16,18,30,31]. Predicting the risk of miscarriage with the size of haematoma is controversial because of the mixed evidence. It has been suggested that size is not the best indicator of the extent of subchorionic bleeding [24]. This is because a larger haematoma can be caused by either an increase in subchorionic bleeding and a decrease in cervical drainage as PV bleeding [24]. This may explain why several studies did not find the size to be predictive of poor prognosis. While size may not correlate linearly with increased risk of miscarriage, haematoma above a certain volume may still confer a higher risk. This is because regardless of the cause, a larger haematoma can have more direct pressure-volume effect on the pregnancy. A significantly larger haematoma may also have greater placental involvement. Currently, the size of haematoma remains a controversial predictor of poor outcome.
Location of haematoma
Haematomas in certain locations may have a worse prognosis. Most SCH are located on the anterior aspect of the uterus and at the peripheries of the placenta [5,17]. Haematomas that were retroplacental or fundal had significantly higher rates of pregnancy loss according to several studies [14,29,30]. A fundal haematoma was four times more likely to cause a miscarriage compared to supra-cervical haematoma (27.5% versus 6.6%) [14]. While retroplacental location was a significant risk factor, Nyberg et al. [29] found that ultimately, it was the degree of placental involvement that best predicted foetal mortality. Using a multiple logistic regression analysis, Nyberg et al. [29] showed that the location was no longer significantly associated with foetal mortality after adjusting for placental involvement. Without any placental involvement, foetal mortality was only 8% [29]. When 20 – 50% of the placenta was involved, foetal mortality climbed to 20%. Greater than 50% of placental involvement resulted in a 75% foetal mortality rate [29]. Based on the current evidence, greater placental involvement and retroplacental or fundal location of haematoma may all be important risk factors for the poorer prognosis amongst women with SCH.
Persistency of haematoma
Most SCH will self-resolve in the second trimester but some can remain symptomatic until the delivery. This persistent SCH is rare and was only present in 0.46% of the general obstetric population [11]. Persistent SCH may carry a worse prognosis. Seki et al. [11] studied 22 cases of persistent SCH and found that while the miscarriage rate was not particularly high (13.6%), most women with persistent SCH experienced preterm labor (77.3%), half of which occurred before 32 weeks. There was also a high prevalence of chorioamnionitis amongst women with persistent SCH (27.3%) [11]. Aoki et al. also found that there is a higher rate of complications, including PTD, SGA, and neonatal lung disease in ten cases of persistent SCH [32]. Although there were no other studies on haematoma that persisted until delivery, several studies observed that haematoma that was slow to resolve or was associated with prolonged PV bleeding had higher rates of pregnancy loss [1,10,23,27]. The evidence is limited but persistent SCH appears to be associated with higher complication rates.
Gestational age of diagnosis
An earlier gestational age of diagnosis of SCH has been found to be a risk factor for worse outcomes in several studies. From a 2005 observational study, SCH diagnosed before 9 weeks has a significantly higher risk of pregnancy loss and an adverse outcome compared to SCH diagnosed after 9 weeks (aOR = 18.29, 95% CI 2.36 – 41.46 and aOR = 2.22, 95% CI 1.13 – 4.40, respectively), even after adjusting for other factors such as haematoma size and maternal age [33]. The study also showed that the risk of pregnancy loss increased from less than 2% to 20%, if the diagnosis occurred before 9 weeks [33]. Similar results were obtained by Bennett et al. [26]. However, the studies were not clear on when the symptoms of PV bleeding occurred in relation to the diagnosis of SCH. Furthermore, many other studies did not agree that an earlier gestational age of diagnosis was a significant prognostic factor [16,27,29,34]. In one study, an earlier gestational age of diagnosis was strongly associated with preterm labor but not pregnancy loss [29]. Yet, according to a 2003 study, there was no significant correlation between gestational age of diagnosis and risk of PTD [34]. Based on the current evidence, gestational age of diagnosis is not a clear risk factor for worse outcomes. A probable mechanism on how an earlier gestational age of diagnosis leads to adverse outcomes is also lacking in the existing studies.
Severity of symptoms
Asymptomatic SCH are common and benign [13,23,35]. In women with SCH, those that experienced PV bleeding were more likely to have PTD than those who were asymptomatic (OR = 4.8, 95% CI 1.2 – 15.9) [34]. Based on a study by Abu-Yousef et al. [27], most women (83%) with moderate-to-heavy PV bleeding had an unfavorable outcome. In contrast, most women (75%) with light PV bleeding had a favorable outcome [27]. The risk of an adverse pregnancy outcome is higher with more severe symptoms in SCH.
Management
Despite the effects SCH might have on pregnancy, there is no specific management guideline for SCH. This is partly because there are only limited studies on how to manage SCH. Currently, most women with SCH are regularly monitored using ultrasound until the haematoma resolves. Otherwise, women with SCH are managed similarly to other women with threatened miscarriage, with advice on bed rest and supplementary progestogen. However, bed rest is not considered to be beneficial for women with threatened miscarriage based on the results of a Cochrane review [36]. There was one non-randomised controlled trial that showed a lower miscarriage rate in women with SCH that had bed rest (6.5% versus 23.3%) [37]. However, given that the study lacked randomisation and was performed retrospectively, the evidence is weak and inconclusive. In terms of progestogen, a Cochrane review of four randomised controlled trials found that it reduced the rate of spontaneous abortion in women with threatened miscarriage significantly (RR = 0.53, 95% CI 0.35 – 0.79) [38]. The beneficial effects of progestogen may be related to its immunomodulatory properties. Progestogen increases the production of progesterone-induced blocking factor, which favors T-helper cell type 2 response [39-41]. Besides that, progestogen may also help by promoting implantation and inhibiting uterine contraction and cervical dilation [39,41]. In several trials, progestogen was beneficial for women with SCH [40,41]. Pandian reported that dydrogesterone given as 40 mg/day stat. followed by 10 mg twice daily until 16 weeks gestation reduced the miscarriage rate by 15.9% (OR = 0.36, 95% CI 0.172 – 0.756) [41]. In another study, taking 40 mg of oral dydrogesterone daily until 16 weeks gestation resulted in maintenance of pregnancies for 93% of women with SCH [40]. Although the results are encouraging, more studies are needed to confirm the benefits of progestogen. Patients and clinicians should weigh the cost and benefits carefully, before starting on progestogen treatment.
A novel drug, called vaginal alpha lipoic acid (ALA), is currently being investigated for its potential use in SCH management [42]. A randomised controlled trial has shown that women taking 10 mg of vaginal ALA had faster resorption of SCH compared to women taking 400 mg progesterone and women without any medication [42]. ALA is thought to be beneficial because of its immunomodulatory properties [42]. However, the trial was a small pilot study with only 76 patients [42]. It is not powered to detect a change in the clinical outcome of miscarriage rate (3/27 in ALA group and 6/27 in progesterone group) [42]. It is still unclear whether faster resorption of SCH would improve clinical outcomes.
Learning points for medical students
For women with symptoms of threatened miscarriage, SCH is the most common ultrasound abnormality detected. It has been suggested that the cause of SCH may be poor placentation, which leads to formation of weak marginal uteroplacental veins that tear and bleed. SCH significantly increases the risk of placental abruption but studies are still conflicting on whether it increases the risk of pregnancy loss and other adverse outcomes including PTD, SGA, pre-eclampsia, and chorioamnionitis. Predictors of poor outcomes include the size of haematoma, location with greater placental involvement, persistency of haematoma, earlier gestational age of diagnosis, and severity of symptoms. Management of SCH involves regular ultrasound monitoring. There are potential benefits with bed rest and supplementary progestogen in some studies but the evidence is still limited. Vaginal ALA is a novel treatment option that is still under investigation. In the future, larger controlled studies that measure all the various prognostic factors will help provide better information on the risk posed by SCH.
Acknowledgements
I would like to thank Dr. Shavi Fernando for his advice and the Monash Diagnostic Imaging Department for providing the ultrasound image of the subchorionic haematoma.
Conflicts of interest
None declared
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Aim: To ascertain the function of miRNA-34a in hepatocellular carcinoma (HCC) and to assess its use as a therapeutic agent through the analysis of pre-clinical and clinical trials.
Discussion: Multiple studies found that miRNA-34a was down-regulated in the majority of human HCC samples and subsequently had a tumour suppressor role via the inhibition of a number of target genes essential for carcinogenesis. MRX34, a miRNA-34a mimic, is currently in an ongoing phase I clinical trial. Interim data has indicated that this therapy has a manageable safety profile, with a partial response observed in one patient. The combination of miRNA-34a with other agents has also proven to exert enhanced anti-tumour effects. Conversely, many studies have reported that miRNA-34a was up-regulated in HCC samples, particularly in those with activation of the beta-catenin pathway.
Conclusion: Pre-clinical studies have shown promising results in the use of a miRNA-34a mimic in HCC as a single agent or as a combination therapy, however, the results from the phase I trial are yet to be fully established. The mechanisms of miRNA-34a in HCC remain to be elucidated, with further research required into its proposed oncogenic role, especially relating to the clinical implications of this interaction.
Introduction
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths worldwide and is the most common form of liver cancer, accounting for between 85% and 90% of primary liver cancers [1]. The major risk factors for hepatocellular carcinoma include hepatitis B (HBV) or C virus (HCV) infection, smoking, alcohol, and aflatoxin [2]. Currently the prognosis for HCC is poor with Australian statistics indicating that the five year relative survival rate for primary liver cancers is only 16% [3]. The non-specific tyrosine kinase inhibitor sorafenib currently represents the only effective treatment against HCC [4]. This poor prognostic outlook and the limited availability of targeted molecular agents for HCC has led to the development of new therapies such as microRNAs (miRNAs).
miRNAs are short (19-24 nucleotides), non-coding RNA molecules that are post-transcriptional regulators of gene expression. Initially, miRNA is transcribed as primary miRNA (pri-miRNA), which is processed into precursor miRNA (pre-miRNA). This is then transported from the nucleus into the cytoplasm where it is processed into its mature form by the enzyme Dicer. The mature miRNA forms part of the RNA-induced silencing complex (RISC), which is responsible for regulating the output of protein-coding genes. These miRNAs interact with the 3’ untranslated regions (UTRs) of the protein-coding genes to result in a decrease in protein output via mRNA degradation or translational repression [5]. Alternatively, miRNAs can result in post-transcriptional stimulation of gene expression via a multitude of direct and indirect mechanisms [6]. It is estimated that a single miRNA can target hundreds to over one thousand different mRNAs, ultimately resulting in miRNAs being responsible for the regulation of around 20-30% of all protein-encoding genes [7]. These miRNAs have been reported to have key roles in cancer initiation, progression, and metastasis [8]. Oncogenic miRNAs are miRNAs that are up-regulated in cancer cells and promote carcinogenesis via the inhibition of tumour suppressor genes. Conversely, the miRNAs that are decreased in cancer cells are known as tumour suppressor miRNAs, as they normally inhibit proto-oncogenes to prevent cancer from developing [9]. The mammalian miRNA-34 family consists of miRNA-34a, which is encoded via its own individual transcript, and miRNA-34b and miRNA-34c, which possess a common primary transcript [10]. Due to the promising and extensive research conducted on miRNA-34a, this review article focused specifically on this particular isoform. Dysregulation of miRNA-34a has been implicated in a wide variety of cancers, including prostate, colon [11], and HCC [12]. The purpose of this review is to analyse the specific role of miRNA-34a in HCC, including addressing contradictory findings and investigating the recent clinical trials.
Table 1. Percentage of human HCC samples with decreased miRNA-34a expression compared to surrounding non-cancerous liver tissue.
The tumour suppressor role of miRNA-34a in hepatocellular carcinoma
A range of studies established that in the majority of human HCC samples, miRNA-34a expression was decreased in comparison to the surrounding non-cancerous liver tissue (Table 1) [12-16]. A murine model of hepatocarcinogenesis induced by a methyl-deficient diet also resulted in the down-regulation of miRNA-34a [17]. Low expression of miRNA-34a in HCC samples has been correlated with a shorter overall [13-15] and disease-free survival [14], as well as higher recurrence rates [13] when compared with samples that displayed up-regulation of miRNA-34a. The decreased expression of miRNA-34a is thought to be caused by genetic alterations such as deletions, point mutations, or chromosomal translocations of its genomic region 1p36 [18], which is common in HCC [19, 20]. Alternatively, this decreased expression has been linked to inactivating mutations of the p53 gene [18], as the induction of miRNA-34a is correlated with p53 status [21]. Furthermore, epigenetic silencing of miRNA-34a has been implicated with these decreased expression levels in multiple forms of cancer, via abnormal CpG methylation in its promoter region [22].
The administration of a miRNA-34a mimic (MRX34) has been shown to cause inhibition of a number of genes within multiple oncogenic pathways such as Wnt/ beta-catenin, c-MET, VEGF, hedgehog, and MAPK (all of which have been implicated in hepatocarcinogenesis), as well as stimulating multiple genes of the p53 pathway [23]. Daige et al. explored a diverse range of HCC related pathways, demonstrating how miRNA-34a exerts its anti-cancer effects by modulation of a number of genes responsible for processes such as metastasis, cellular proliferation, cell cycle regulation, apoptosis, and cellular senescence [23].
miRNA-34a and cellular proliferation, cell cycle regulation, and apoptosis
A number of cell culture studies investigated the effects of ectopic expression of miRNA-34a in the HepG2 cell line, with contradicting results [12,16,18]. Ectopic expression of miRNA-34a caused significant inhibition of cellular proliferation at 72 hours [18] and 96 hours [16] post- transfection. In addition, miRNA-34a was demonstrated to regulate the cell cycle via inducing G1 arrest [18]. Furthermore, it has been found that miRNA-34a can induce apoptosis, as determined by increased caspase 3/7 activity [16]. In contrast, other reports claimed that there was no effect on cellular proliferation, G1 arrest, or apoptosis, [12,18], highlighting the conflicting information within the current literature. The discrepancies between these three studies could partially be explained by the varying methods used to express miRNA-34a, and the different measurement times post-transfection (48, 72 or 96 hours) [16]. Additional research has found that miRNA-34a induces apoptosis in HCC cells via binding to the 3’ UTR of the Bcl-2 mRNA, causing inhibition of its translation [15]. Over-expression of miRNA-34a has also been correlated with a decreased expression of Bcl-2 in a number of other HCC studies [14,24].
miRNA-34a and metastasis
Down-regulation of miRNA-34a expression has been associated with metastatic HCC [12,16,25]. Multiple studies have established that the ectopic[1] expression of miRNA-34a in the HepG2 cell line inhibits tumour cell migration and invasion via silencing of the c-Met gene, which subsequently decreases the c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) [12,16]. Cheng et al. also proposed that miRNA-34a prevents tumour migration, invasion, and metastasis by decreasing cathepsin D [18], a protease that contributes to the degradation of the basement membrane [26]. Furthermore in the HCC cell line Hca-F (high lymphatic metastatic potential) it was determined through in vitro and in vivo studies in mice that the ectopic expression of miRNA-34a caused a reduction in the metastatic potential [27].
miRNA-34a and cellular senescence
Earlier studies showed that miRNA-34a induced cellular senescence via cell cycle arrest in pathways that were telomere-independent [13, 28]. Recently, miRNA-34a over-expression has been shown to induce senescence in HCC cells in a telomere-dependent manner, regulated by p53. This cellular senescence occurs by the inhibition of FoxM1 and c-Myc, which causes the inactivation of telomerase activity, resulting in telomere shortening (Figure 1) [13].
Figure 1. Telomere dependent pathway of miRNA-34a induced cellular senescence (adapted from Xu et al. [13]). High levels of miRNA-34a, which is regulated by p53, results in the inhibition of FoxM1 and c-Myc, causing the inactivation of telomerase, leading to telomere shortening and subsequently cellular senescence.
miRNA-34a delivery methods
There is a vast array of delivery systems, both viral and non-viral, that are used to increase miRNA expression. Both mechanisms are associated with advantages and disadvantages [29]. Rubone and MRX34 are two prominent non-viral delivery methods that have been investigated in HCC studies [23,24]. Rubone is a small molecular modulator of miRNA-34a that was shown to induce miRNA-34a expression selectively in HCC cells (although only those with wild-type or mutant p53, not with p53 deletion), causing inhibition of tumour growth both in vitro and in vivo in the HepG2 xenograft mouse model. Xiao et al. also found that this miRNA-34a modulator displayed similar or even greater anti-HCC activity than sorafenib, the current treatment for advanced HCC [24]. MRX34 is a double-stranded miRNA-34a mimic that is delivered by liposomes [30]. The systemic delivery of this molecule resulted in tumour regression during in vivo studies in two different xenograft mouse models (Hep3B and HuH7) of liver cancer [23]. An oncolytic adenoviral vector that co-expressed miRNA-34a and IL-24 has also been studied in a HCC model. This was found to cause increased anti-tumour activity both in vitro and in vivo, predominantly via the downregulation of SIRT1 and Bcl-2 [14].
Clinical trials
Currently there is an ongoing phase I trial of MRX34 [31], which commenced in April 2013 and was originally indicated for patients with primary liver cancer or cancers with metastasis to the liver [32]. It was then gradually expanded to include patients with other advanced solid tumours (with or without liver metastasis) and haematological malignancies (lymphoma and multiple myeloma) [31]. There were 75 patients with advanced solid tumours enrolled in this study and 30 of these had HCC [33]. This trial’s data revealed that partial responses to the treatment, as per the RECIST guidelines (Table 2) [34], were observed in one patient with HCC, one with melanoma, [33] and one with advanced renal cell carcinoma [35]. Furthermore a number of patients in this trial were found to have attained a stable disease state [35]. This interim phase I data also determined that MRX34 has a manageable safety profile [33]. A recent press release from MIRNA therapeutics has indicated that phase II clinical trials will commence by the end of 2016 and will consist of two studies, one on renal cell carcinoma and the other on melanoma [35]. In terms of HCC-specific trials with MRX34, the future direction is currently unclear and with limited data available at the present time it is difficult to draw any definitive conclusions.
Table 2. Revised RECIST guidelines [34]
Combination therapy
Yang et al. demonstrated that ectopic expression of miRNA-34a resulted in the sensitisation of HCC cells to sorafenib-induced apoptosis and toxicity via inhibiting expression of Bcl-2 [15]. Additionally, the administration of miRNA-34a was found to sensitise HCC cells to chemotherapy (cisplatin) in vitro through the AXL pathway [36]. The combination of a miRNA-34a mimic and C-met inhibitor also resulted in a greater inhibition of cell growth and induction of apoptosis in vitro than either of these two therapies alone [16]. However, to establish more definitive results, further research is required in this field, particularly in regards to clinical trials.
The oncogenic role of miRNA-34a in hepatocellular carcinoma
Conversely, a number of other studies have shown increased expression of miRNA-34a in both murine and human HCC tissues [37-41], suggesting it may have an oncogenic role in addition to its tumour suppressor role. A recent article has investigated these claims and found that miRNA-34a displays oncogenic properties in liver tumours with beta-catenin activation [40]. Increased beta-catenin activation is most commonly caused by mutations in the CTNNB1 gene (the gene encoding beta-catenin), and this is estimated to occur in 20-40% of HCCs [42]. Gougelet and colleagues demonstrated using ApcKO mice (ApcKO causes activation of the Wnt/beta-catenin pathway [43]) that administration of a miRNA-34a inhibitor (LNA-34a) caused increased expression of hepatocyte nuclear factor 4a. This leads to increased apoptosis predominantly via caspase 2 activation and decreased cell proliferation through inhibition of cyclin D1 (Figure 2). This theory was then postulated to complement the data of the studies listed in Table 1, by accounting for those samples that showed up-regulation of miRNA-34a expression. The disparity in results between studies is thought to be due to the varying causes of HCC [40]. For example, the majority of HCC samples from the studies in the Table 1 were HBV+, and this was found to be associated with a lower frequency of CTNNB1 mutations [44]. Conversely HCC resulting from HCV infection has been shown to have a higher rate of CTNNB1 mutations [45]. However, these findings contradict the theory that miRNA-34a has a tumour suppressor function, and with relatively limited research on this oncogenic pathway, further investigation is required. Studies investigating the concept of miRNA-34a having a tumour suppressor or oncogenic function depending on the cause of the tumour would also be important, as well as an investigation of the clinical implications of this relationship.
Figure 2. Role of miRNA-34a inhibitor in counteracting the oncogenic action of miRNA-34a in HCC with beta-Catenin activation (Adapted from Gougelet et al. [40]). The miRNA-34a inhibitor LNA-34a causes a reduction in β-catenin induced miRNA-34a expression which leads to an increased expression of HNF-4α. Subsequently cyclin D1 levels are reduced, leading to decreased cell proliferation and increased caspase 2 levels, leading to increased apoptosis. The combined effects of these two actions then leads to the decreased progression of the hepatocellular carcinoma.
Limitations
A number of limitations were identified within this review, particularly relating to the conflicting information and the limited availability of clinical trial results. Contradictory information was noted on a number of occasions, especially with the use of a miRNA-34a inhibitor for HCC, making it difficult to evaluate a clear clinical benefit to this potential therapy. The data surrounding the clinical trial was also restricted as the trial is ongoing. Subsequently, all the data had to be sourced from press releases and abstracts from presentations at conferences, which were all funded by MIRNA therapeutics; thus a potential conflict of interest was noted.
Conclusion
Results have indicated that miRNA-34a has a tumour suppressor function in HCC and is responsible for the down-regulation of a number of genes involved in carcinogenesis. There is, however, contradicting information described in studies investigating these parameters, highlighting the complexity of this topic. Rubone and MRX34 are two prominent miRNA-34a delivery systems that were shown to exert anti-tumour activity in pre-clinical models. Additionally, MRX34 has been commenced in a clinical phase I trial that is still currently ongoing, with a partial response already observed in one patient. However, the future of HCC-specific MRX34 trials remains unclear as limited information is currently available. Based on the promising results of miRNA-34a as a combination therapy, this is an area that requires further investigation through clinical trials. Conversely, other sources have found that miRNA-34a plays an oncogenic role in HCC, particularly in those with beta-catenin activation. Subsequently, it was demonstrated that miRNA-34a inhibitors should be used in these instances. Further research is necessary in order to ascertain the clinical implications of using a miRNA-34a mimic or inhibitor depending on the beta-catenin mutation status of the patient.
Conflicts of interest
None declared.
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