Primary hepatic tumours are one of the most aggressive and resistant forms of cancer. Early diagnosis of liver cancer and the development of more accurate markers for biological classification are crucial to improving the clinical management and survival of patients. This article discusses the emerging use of microRNAs for the diagnosis of liver cancer.

by Dr Luc Gailhouste and Dr Takahiro Ochiya

Liver cancer and diagnosis
Primary liver cancer is mainly represented by hepatocellular carcinoma (HCC) and accounts for almost 90% of primitive hepatic malignancies. Statistically, HCC is the third most common cause of death from cancer worldwide [1] and is generally encountered in patients exhibiting an underlying chronic liver disease such as hepatitis B virus (HBV) and/or C virus (HCV) infection, alcohol abuse, or liver steatosis. Chronic hepatitis leads to fibrosis and gradually evolves into cirrhosis. Global studies estimate that approximately 80–90% of all HCCs arise from cirrhotic livers. Despite great advances in the treatment of the disease, hepatic cancer exhibits one of the lowest remission rates (less than 10% after five years), mainly due to its late diagnosis and high resistance to the conventional agents of chemotherapy. Indeed, as such a disease tends to remain asymptomatic, approximately 50% of newly diagnosed patients already exhibit late advancement.

Common HCC diagnostic methods include liver imaging techniques such as triphasic computed tomography scanning, magnetic resonance imaging (MRI), and abdominal ultrasound [2]. A panel of serological biochemical markers, including aminotransferases ALAT and ASAT, has also been used for several decades to monitor liver pathologies in a non-invasive manner.

Until recently, imaging tests were frequently combined with the non-invasive measurement of serum alpha-fetoprotein (AFP). Normally produced by the fetal liver, AFP decreases soon after birth whereas its high level in adults can be correlated with the appearance of malignant hepatic disease. However, the American Association for the Study of Liver Diseases (AASLD), in its practice guidelines, discontinued the use of the blood tumour marker AFP for surveillance and diagnosis due to the limited sensitivity and specificity of the method. When uncertainty regarding the diagnosis persists, a percutaneous biopsy followed by histological examination of the nodule is indicated [3]. This technique remains the gold standard method for determining the degree of underlying fibrosis and shows appreciable sensitivity (more than 80%) for HCC diagnosis.

An important breakthrough in the clinical management of liver cancer would come from the accurate correlation of the alterations of cancer-related genes and the tumour phenotype. Although HCC lesions can be broadly distinguished by histological or immunological assessment, their prognosis and clinical evolution vary greatly from one individual to another. The discovery of innovative and effective biomarkers ensuring an early diagnosis of the disease correlated with the etiology, the pathogenic tendency, and the malignancy of the tumour could significantly enhance the molecular assessment of HCC and its classification in order to maximize the positive response of therapeutics.

MicroRNAs: biogenesis and mechanism of action
MicroRNAs (miRNAs) constitute a group of evolutionary conserved small non-coding RNAs of approximately 22 nucleotides that accurately regulate gene expression by complementary base pairing with the 3’-untranslated regions (3’-UTRs) of messenger RNAs (mRNAs) [4]. These post-transcriptional regulators were first evidenced in C. elegans by Ambros and co-workers who discovered that lin-4, a gene known to control the timing of nematode larval development, did not code for a protein but produced small RNAs that specifically bind to lin-14 mRNA and repress its translation.

miRNA biogenesis is a multistep process that has been reviewed extensively [Figure 1]. An essential feature of miRNAs is that a single miRNA can recognize numerous mRNAs, and, conversely, one mRNA can be recognized by several miRNAs. These pleiotropic properties enable miRNAs to exert wide control over a plethora of targets, attesting to the complexity of this mechanism of gene expression regulation. Several reports have described the key role of these post-transcriptional regulators in the control of diverse biological processes such as development, differentiation, cell proliferation, and apoptosis. The alterations of miRNA expression have also been reported in a wide range of human diseases, including cancer [5].

In HCC, the atypical expression of miRNAs frequently contributes to the deregulation of critical genes known to play an essential role in tumorigenesis and cancer progression. The current consensus is that cancer-related miRNAs function as oncogenes or tumour suppressors [6]. As for other malignancies, two situations can occur in HCC: (i) tumour suppressor miRNAs can be downregulated in liver cancer and cause the upregulation of oncogenic target genes repressed in normal hepatic tissues, increasing cell growth, invasion abilities, or drug resistance; (ii) oncogenic miRNAs, also called oncomirs, can be upregulated in HCC and can downregulate their target tumour suppressor genes, potentially leading to hepatocarcinogenesis.

miRNA as a diagnostic tool
As miRNA signatures are believed to serve as accurate molecular biomarkers for the clinical classification of HCC tumours, the availability of consistent technologies that enable the detection of miRNAs has become of interest for both fundamental and clinical purposes. The most current detection methods commonly used are microarray and real-time quantitative polymerase chain reaction (RT-qPCR).

Microarray analysis presents the advantage of offering a high speed of screening by employing various miRNA probes within a single microchip. However, the technique has lower sensitivity and specificity than RT-qPCR, which is the most widely used method.

miRNA RT-qPCR is based on the use of stem–loop primers, which can specifically bind to the mature miRNA during reverse transcription, granting a high degree of accuracy to the method [7]. Analysis of miRNAs by RT-qPCR is a cost-effective technique and, due to its efficiency, a valuable way to validate miRNA signatures. Moreover, the development of RT-qPCR protocols has improved the sensitivity of miRNA detection down to a few nanograms of total RNAs. This amount can be easily and routinely obtained by extracting total RNAs from a small fragment of a hepatic percutaneous biopsy.

A plethora of studies have already reported various miRNA profiles potentially reflecting HCC initiation and progression that could be employed as specific cancer biomarkers [8]. Comparative analysis of bibliographic data provides evidence of the persistent augmentation of miR-21 in cancer, regardless of the tumour origin. In the HCC, miR-21 is also frequently overexpressed where it acts as an oncogenic miRNA. The major overexpression of miR-21 is associated with the inhibition of the tumour suppressor PTEN and the poor differentiation of the tumour. The use of an miRNA-based classification correlated with the etiology and the aggressiveness of the tumour appears very promising, as it could significantly enhance the accuracy of the molecular diagnosis of HCC and its classification, leading to the consideration of more appropriate therapeutic strategies.

In this regard, Budhu and collaborators defined a combination of 20 miRNAs as an HCC metastasis signature and showed that this 20-miRNA-based profile was capable of predicting the survival and recurrence of HCC in patients with multinodular or single tumours, including those at an early stage of the disease [9]. Remarkably, the highlighted expression profile showed a similar accuracy regarding patient prognosis when compared to the conventional clinical parameters, suggesting the relevance of this miRNA signature. Consequently, the profiling of aberrantly expressed cancer-related miRNAs might establish the basis for the development of a rational system of classification in order to refine the diagnosis and the prediction of HCC evolution.

Tumour suppressor miRNA: the case of miR-122
The case of miR-122 is of prime interest, first, because it represents by itself more than half of the total amount of miRNAs expressed in the liver [10]. Remarkably, miR-122 is a key host factor required for HCV replication. A phase 2 clinical trial was recently initiated that reported the world’s first miRNA-based therapy targeting miR-122 in HCV-infected patients using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen [11]. Thus, a four-week miravirsen treatment by subcutaneous injection provided long-lasting antiviral activity and was well tolerated.

However, the experimental silencing of miR-122 resulted in increased expression of hundreds of genes normally repressed in normal hepatocytes. The miR-122 knockout mouse model displays hepatosteatosis, fibrosis, and a high incidence of HCC, suggesting the tumour suppressor role of miR-122 in the liver. In primary liver carcinoma, the existence of an inverse correlation was demonstrated between the expression of miR-122 and cyclin G1, which is highly implicated in cell cycle progression.

Regarding the potential of miR-122 as a diagnostic biomarker in liver cancer, numerous studies have already reported the significant and specific downregulation of miR-122 expression in both human and rodent HCC models. Obviously, miR-122 was shown as downregulated in more than 70% of the samples obtained from HCC patients with underlying cirrhosis as well as in 100% of the HCC-derived cell lines [12].

To illustrate this statement, we analyzed the expression levels of miR-122 in 20 patients who exhibited HCC using RT-qPCR. Following RNA extraction from biopsies with the miRNeasy Mini Kit (Qiagen), 100 ng of total RNA was reverse-transcribed using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems). The expression levels of mature miR-122 were determined in each sample by RT-qPCR with Taqman Universal PCR Master Mix in a 7300 Real-Time PCR System from Applied Biosystems. The expression levels of miRNAs were normalized with respect to the endogenous levels of RNU6B. RT-qPCR data were obtained easily and rapidly by a routinely conventional method used in our laboratory. As a result, miR-122 expression was reduced more than threefold in HCC biopsies relative to the normal liver group (median 0.935 and 3.495, respectively; P<0.0001, Mann–Whitney U test) [Figure 2]. These data suggest that cancer-related miRNAs, such as miR-122, which are deregulated in HCC tissues, could be relevant with regard to the development of new diagnostic tools and the clinical management of liver cancer patients. Conclusions and emerging approaches
The expression profile of specific miRNAs has been found to reflect the biological behaviour of HCC tumours, such as aggressiveness, invasiveness, or drug resistance. As a consequence, miRNA investigations may offer opportunities to determine miRNA signatures that would provide valuable information to stratify and refine HCC diagnosis in terms of prognosis, response to treatment, and disease relapse. Recently, tumour-derived miRNAs have been efficiently detected in the serum of patients and characterized as potential non-invasive biomarkers for HCC.

The concept that miRNAs could serve as potential plasma markers for liver diseases is, thus, gaining attention. Due to its frequent deregulation in viral hepatitis, cirrhosis, and cancer as well as its specific and massive expression in the liver, the assessment of serum miR-122 could represent one reliable strategy for the non-invasive diagnosis of chronic liver pathologies. Although the process of assessing serum miRNAs remains under improvement, cancer-related circulating miRNAs represent an exciting and promising field of investigation for the development of more accurate technologies for the early diagnosis of HCC.

References
1. Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006; 6: 674–687.
2. Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002; 122: 1609–1619.
3. Ryder SD. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 2003; 52(Suppl 3): iii1–8.
4. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
5. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857–866.
6. Gailhouste L, Ochiya T. Cancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma. Histol Histopathol 2012.
7. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.
8. Gailhouste L, Gomez-Santos L, Ochiya T. Potential applications of miRNAs as diagnostic and prognostic markers in liver cancer. Front Biosci 2013; 18: 199–223.
9. Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47: 897–907.
10. Girard M, Jacquemin E, Munnich A, et al. miR-122, a paradigm for the role of microRNAs in the liver. J Hepatol 2008; 48: 648–656.
11. Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol 2012; 199: 407–412.
12. Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67: 6092–6099.

The authors
Luc Gailhouste PhD and
Takahiro Ochiya PhD

Division of Molecular and Cellular
Medicine, National Cancer Center Research Institute, Tokyo, Japan

Plasma levels of heart-type fatty acid binding protein (H-FABP) have been shown to rise early after the onset of acute myocardial infarction (AMI). Recent evidence suggests combining H-FABP with troponin gives superior diagnostic accuracy compared to the alternative ‘early markers’ of myocardial necrosis, creatine kinase-MB (CK-MB) and myoglobin. However, using a single measurement at the time of presentation to the Emergency Department (ED), H-FABP is unlikely to have sufficient sensitivity to safely ‘rule out’ AMI, even when combined with a standard troponin assay. With the advent of high sensitivity troponin assays which have higher diagnostic sensitivity at the time of presentation, it is possible that H-FABP could be combined with levels of high sensitivity troponin and potentially with other clinical information to enable safe ‘rule out’ of AMI using a single blood test at the time of presentation. Further work in this area is needed.

by Dr Richard Body

Background
Suspected cardiac chest pain accounts for approximately one quarter of acute medical admissions, although only a minority of the patients admitted will ultimately be diagnosed with an acute coronary syndrome [1]. Meanwhile, up to 2% of patients with acute myocardial infarction (AMI) have that diagnosis missed and are inadvertently discharged, leading to a worse prognosis [2]. There is therefore tremendous potential to reduce unnecessary hospital admissions in this patient group, although advances in diagnostic technology are clearly necessary in order to do so.

High sensitivity troponin
Cardiac troponins are regulatory proteins contained within the myofibrillar apparatus of cardiac myocytes. They are released into the bloodstream following myocardial necrosis and their detection allows highly sensitive and specific diagnosis of AMI. Indeed, the detection of a rise and/or fall of cardiac troponin in serum or plasma is integral to the diagnosis of AMI. With the advent of high sensitivity troponin (hs-cTn) assays, which have greater analytical and diagnostic sensitivity than standard assays, it is tempting to believe that the hunt for an ‘early rule out’ strategy for acute coronary syndromes is over. Standard troponin assays lack the diagnostic sensitivity to enable safe exclusion of acute myocardial infarction (AMI) when measured at the time of presentation. This creates a period of ‘troponin blindness’, when patients with AMI still have low circulating troponin levels prior to the development of a late troponin rise. Hs-cTn assays have been shown to improve diagnostic sensitivity at the time of initial presentation to the Emergency Department (ED). While this reduces the magnitude of our problem with ‘troponin blindness’, it does not overcome the problem completely. Even hs-cTn assays fail to identify approximately 10% of patients with AMI at the time of presentation [3, 4]. With hs-cTn assays it may be possible to reduce the time taken to confidently ‘rule out’ AMI with serial sampling from 6 to 9 hours after arrival (or 10–12 hours from symptom onset) to as little as 3 hours after arrival [4, 5]. This approach still needs to be validated against a hs-cTn reference standard, however, and there are a few other reasons to be cautious. The sensitivity of the Siemens troponin I Ultra assay (a sensitive assay but not high sensitivity), which had a diagnostic sensitivity of 100% at 3 hours after presentation in Keller et al.’s original study (evaluated against the reference standard of testing 6 hours after arrival), was actually only 94.5% at 6 to 12 hours from symptom onset [4]. Further, high sensitivity troponin T (hs-cTnT) has been shown to have a sensitivity of only 92.2% when measured 2 hours after presentation, which is still some way from a satisfactory rule out strategy [6]. Using the new Abbott Architect high sensitivity troponin I assay, sensitivity for AMI is 98.2% (with 95% confidence intervals extending down to 96.9%), again using a standard troponin assay as the reference standard [5]. Even if we accept that no rule out strategy will be 100% sensitive and consider this 3-hour troponin to be a satisfactory rule out strategy, that still means an anxious wait for patients and would still, in health systems like the United Kingdom, necessitate admission to an inpatient ward for investigation.

Interest in ‘early markers’ of myocardial necrosis
There has been interest in the role of ‘early markers’ of myocardial necrosis for many years. As troponin is predominantly an intracellular constituent and levels do not peak for 12 to 24 hours after the onset of infarction [7], many have investigated the value of biomarkers with release kinetics suggesting that they may enable earlier identification of AMI. Thus, the measurement of creatine kinase-MB (CK-MB) and myoglobin levels in combination with troponin were shown to improve early diagnosis of AMI as early as 2001 [8]. More recently, the ASPECT study from 14 countries in the Asia-Pacific region examined the value of CK-MB, myoglobin and troponin I (using assays from Alere, San Diego, CA, USA) measured at presentation and 120 minutes later in patients with a Thrombolysis In Myocardial Infarction (TIMI) score of 0/7. The authors found that 9.8% of patients could be discharged using this strategy with a 0.9% incidence of adverse cardiac events within 30 days [9]. Around the same time, the Randomised Assessment Using Panel Assay of Cardiac Biomarkers (RATPAC) study demonstrated that serial evaluation of CK-MB, myoglobin and troponin I over 90 minutes led to an increase in the proportion of patients successfully discharged from the ED, although this came at a cost of rebound-overuse of Coronary Care resources, perhaps as a function of the lack of specificity of myoglobin and CK-MB. The strategy was found to be not cost effective [10].

Heart-type fatty acid binding protein
Heart-type fatty acid-binding protein (H-FABP) is a cytosolic protein that is abundantly expressed in human myocardial cells, where it facilitates intracellular fatty acid transport within cardiac myocytes [11]. Plasma H-FABP levels rise early after the onset of AMI. McCann et al. evaluated H-FABP (Hycult Biotechnology ELISA) and troponin T (cTnT; Roche Elecsys, 4th generation) in 415 patients who were admitted to an acute cardiology unit on suspicion of an acute coronary syndrome. They demonstrated that H-FABP had superior sensitivity to troponin in patients who presented early (<4h) after symptom onset [Figure 1] [12]. A meta-analysis of 16 studies including 3,709 patients with suspected AMI demonstrated a pooled sensitivity of 84% [95% confidence intervals (CI) 76–90%] and a pooled specificity of 84% (95% CI 76–89%), although there was significant heterogeneity between studies [13]. It is clear that measurement of H-FABP alone cannot enable safe ‘rule out’ of AMI. Combining H-FABP with troponin will, however, yield a higher diagnostic sensitivity. Body et al. [14] demonstrated that the combination of H-FABP and troponin I offers both superior sensitivity and superior specificity to the combination of CK-MB, myoglobin and troponin I [Figure 2].
A systematic review by Carroll et al. demonstrated that, in 4 studies, the combination of H-FABP and troponin had an overall sensitivity of between 76 and 97% [15]. Two of these studies did, however, use insensitive troponin assays with diagnostic sensitivities of 42% and 55% respectively. The use of more sensitive troponin assays may be expected to yield higher diagnostic performance. Indeed, in the study by Body et al., the sensitivity of the combination of H-FABP and troponin increased from 82% to 87% when a more sensitive troponin assay was used [14, 16]. If only low risk patients (using the modified Goldman risk stratification tool) who had normal H-FABP and normal cTnT were considered for early discharge, a sensitivity and negative predictive value of 99% could be achieved, although this strategy may have a specificity as low as 19%, meaning that only a minority of patients would be eligible for early discharge while 1% of AMIs would still be missed [16].

H-FABP and high sensitivity troponin
It is clear that neither H-FABP nor troponin (even using a high sensitivity assay) can be used to safely exclude a diagnosis of AMI when measured at the time of presentation to the ED. The combination of H-FABP and standard troponin assays improves overall diagnostic sensitivity but is still unable to ‘rule out’ this important diagnosis. By combining H-FABP with high sensitivity troponin assays, it may be possible to further increase sensitivity and thus achieve an effective early rule out strategy. Evidence in this area is still limited. However, Aldous et al. did evaluate the combination of H-FABP (Hycult Biotech) and hs-cTnT in a cohort of 384 patients presenting to the ED with suspected acute coronary syndromes. This combination had a sensitivity of 90.0% for AMI and a specificity of 73.5%. Notably, the sensitivity of the H-FABP assay alone was particularly low in this study (50.0%), which may be a function of the high diagnostic cut-off employed (60ng/ml) when compared to the cut-off employed by McCann et al. using the same assay (5ng/ml) [12, 17]. Using this high diagnostic cut-off, however, the combination of H-FABP and hs-cTnT measured at the time of presentation may help to ‘rule in’ the diagnosis of AMI, with a specificity of 99.4% (95% CI 97.9–99.9%) [17].

Inoue et al. also evaluated both hs-cTnT and H-FABP (DS Pharma Biomedical, Osaka) in 432 ED patients with suspected acute coronary syndromes. In this study, H-FABP had a similar area under the receiver operating characteristic (ROC) curve (AUC) to hs-cTnT (0.83 versus 0.82), although hs-cTnT had a higher sensitivity at the diagnostic cut-off (87.9% vs. 78.5%) [18]. The authors do not report the diagnostic value of the combination of both biomarkers. Meanwhile, in 1,818 patients with suspected acute coronary syndromes, Keller et al. reported that H-FABP had an AUC of 0.89, which rose to 0.97 when combined with high sensitivity troponin I (Abbott Architect STAT high sensitive troponin) [5]. This implies that the combination has high diagnostic accuracy, although the sensitivity and negative predictive value of the strategy were not reported.

H-FABP and prognosis
H-FABP levels may also have prognostic value in patients with suspected acute coronary syndromes. Viswanathan et al. studied 1,080 consecutive patients presenting with suspected acute coronary syndromes [19]. They measured both H-FABP (Randox Evidence Biochip) and troponin I using a sensitive assay (Siemens Advia troponin I Ultra) and followed patients for a median of 18 months. H-FABP predicted death or AMI occurring during follow up, even in troponin negative patients and after adjustment for age and serum creatinine. For predicting death or AMI, H-FABP had an AUC of 0.79 (95% CI 0.74–0.84)
compared to 0.77 (95% CI 0.72–0.82) for troponin I.

Future work
Further work is still needed to determine whether the combination of H-FABP and high sensitivity troponin will enable safe rule out of acute coronary syndromes in the ED. Combination with other clinical information available from risk stratification tools (such as the modified Goldman or TIMI scores) or the ECG may further increase sensitivity, enabling AMI to be safely excluded in a proportion of patients presenting to the ED. Further, with the increase in false positive results given by high sensitivity troponin assays, H-FABP may help to ‘rule in’ the diagnosis of AMI in patients with troponin elevations at the time of presentation, before the results of serial testing are available. This will facilitate early treatment and triage to an appropriate level of care in the hospital, while avoiding the risks of unnecessary treatment for those patients with false positive elevations.

Conclusions
H-FABP is a promising biomarker for use in patients with suspected acute coronary syndromes. Used alone or in combination with a standard troponin assay, sensitivity will be insufficient to safely ‘rule out’ AMI. Further work is needed to determine whether combination with a high sensitivity assay can enable safe ‘rule out’ for a proportion of patients, and to evaluate whether H-FABP may have a role in the differentiation between ‘true positive’ and ‘false positive’ troponin elevations at the time of initial presentation.

References
1. Goodacre S, et al. The health care burden of acute chest pain. Heart 2005; 91: 229–230.
2. Pope JH, et al. Missed diagnoses of acute cardiac ischaemia in the Emergency Department. N Engl J Med 2000; 342: 1163–1170.
3. Reichlin T, et al. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. N Engl J Med 2009; 361: 858–867.
4. Keller T, et al. Sensitive troponin I assay in early diagnosis of acute myocardial infarction. N Engl J Med 2009; 361(9): 868–877.
5. Keller T, et al. Serial changes in highly sensitive troonin I assay and early diagnosis of myocardial infarction. JAMA 2011; 306(24): 2684–2693.
6. Aldous SJ, et al. Diagnostic and prognostic utility of early measurement with high-sensitivity troponin T assay in patients presenting with chest pain. CMAJ 2012; 184: E260-E268.
7. Tucker JF, et al. Early diagnostic efficiency of cardiac troponin I and troponin T for acute myocardial infarction. Acad Emerg Med 1997; 4(1): 13–21.
8. McCord J, et al. Ninety-minute exclusion of acute myocardial infarcdtion by use of quantitative point-of-care testing of myoglobin and troponin I. Circulation 2001; 104: 1483–1488.
9. Than M, et al. A 2-h diagnostic protocol to assess patients with chest pain symptoms in the Asia-Pacific region (ASPECT): a prospective observational validation study. Lancet 2011; 377(9771): 1077–1084.
10. Fitzgerald P, et al, on behalf of the RATPAC investigators. Cost-effectiveness of point-of-care biomarker assessment for suspected myocardial infarction: The RATPAC trial (Randomised Assessment of Treatment Using Panel Assay of Cardiac markers). Acad Emerg Med 2011; 18(5): 488–495.
11. Schaap FG, et al. Impaired Long-Chain Fatty Acid Utilization by Cardiac Myocytes Isolated From Mice Lacking the Heart-Type Fatty Acid Binding Protein Gene. Circ Res 1999; 85(4): 329–337.
12. McCann C, et al. Novel biomarkers in early diagnosis of acute myocardial infarction compared with cardiac troponin T. Eur Heart J 2008; 29(23): 2843–2850.
13. Bruins Slot MH, et al. Heart-type fatty acid-binding protein in the early diagnosis of acute myocardial infarction: a systematic review and meta-analysis. Heart 2010; 96(24): 1957–1963.
14. Body R, et al. A FABP-ulous ‘rule out’ strategy? Heart fatty acid binding protein and troponin for rapid exclusion of acute myocardial infarction. Resuscitation 2011; 82(8): 1041–1046.
15. Carroll C, et al. Heart-type fatty acid binding protein as an early marker for myocardial infarction: systematic review and meta-analysis. Emerg Med J 2012.
16. Body R, et al. Reply to Letter: Still FABP-ulous even with a more sensitive troponin assay. Resuscitation 2012; 83(2): e29–e30.
17. Aldous S, et al. Heart fatty acid binding protein and myoglobin do not improve early rule out of acute myocardial infarction when highly sensitive troponin assays are used. Resuscitation 2012; 83(2): e27–e28.
18. Inoue K, et al. Heart fatty acid-binding protein offers similar diagnostic performance to high-sensitivity troponin T in Emergency Room patients presenting with chest pain. Circulation 2011; 75: 2813–2820.
19. Viswanathan K, et al. Heart-Type Fatty Acid-Binding Protein Predicts Long-Term Mortality and Re-Infarction in Consecutive Patients With Suspected Acute Coronary Syndrome Who Are Troponin-Negative. J Am Coll Cardiol 2010; 55(23): 2590–2598.

The author
Richard Body, MB ChB MRCSEd(A&E) FCEM PhD
Emergency Department,
Manchester Royal Infirmary,
Oxford Road, Manchester, M13 9WL, UK
e-mail: richard.body@manchester.ac.uk

Optical coherence tomography (OCT) has long been routinely used in ophthalmology, but recent studies in the field of renal cell carcinoma have demonstrated the ability of OCT to distinguish between renal malignancies and normal renal tissue. This suggests the possibility that, eventually, diagnosis by invasive biopsy could be replaced by non-invasive techniques.

by D. M. de Bruin, Dr P. Wagstaf, Dr K. Barwari, Prof. T. G. van leeuwen, Dr D. J. Faber, Prof. J. J. de la Rosette and Dr M. P. Laguna

The diagnosis of small renal masses
The diagnosis of small renal masses (SRMs) has seen a dramatic increase in presentation in recent decades. This change is mainly attributed to an increased use of abdominal imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI). However, the large imaging depth of such modalities is accompanied by a relatively low resolution of the obtained images, hindering conclusions at the level of histological composition. Recent studies have shown an inverse correlation between tumour size and malignancy, and up to 10 % of all extirpated (and thus deemed malignant) tumours appear to be benign on histopathological examination. This inverse relationship increases to 25% when small renal masses (SRM) (≤4 cm) are considered [1]. Therefore, pre-operative diagnosis of (small) renal tumours would be desirable. However, due to the high number of non-diagnostic biopsy results (up to 30 % in SRM), systematic use of pre-operative renal mass biopsies is still not recommended in the major guidelines [2–5].

Renal mass biopsy
Most renal biopsies are performed percutaneously and are supported by image guidance using computed tomography (CT) or ultrasound. The biopsies are normally performed under local anesthesia in an outpatient setting. When a renal  tumour is evaluated, a biopsy can deliver one of two results: diagnostic (benign or malignant) or non-diagnostic, the later including the presence of necrosis, fibrosis and normal renal parenchyma with absence of  tumour cells [Figure 1]. When the biopsy is diagnostic, other characteristics such as histopathologic subtype and grade can also be assessed [4, 6, 7].

Conceptually a failed biopsy means that there is no  tumour tissue available for assessment in the biopsy specimen, although other types of tissue might be present in the sample. The reason for a failed biopsy could be a technical failure of the puncture method (e.g. misfire or malfunctioning of the biopsy gun) or incorrect sampling caused by imperfect image guidance. Incorrect sampling is sometimes unavoidable due to the nature of renal  tumours, which may contain necrotic and fibrotic tissue, or be mixed in nature with solid and cystic components. Also, the presence of normal renal tissue implies that the sampling is incorrect, as very few renal masses are composed of normal renal tissue. The presence of fibrotic, inflammatory, fatty or necrotic tissue in the specimen means that a differential diagnosis between malignant and benign tumour cannot be made. Besides the fact that histopathological analysis requires time, it is also subject to a certain degree of discordance among different pathologists [8].

A diagnostic imaging tool that allowed real-time visualization of micro-scale tissue architecture and subsequent differentiation of tissue type during the procedure would accelerate and simplify the overall diagnostic procedure.

Optical imaging
Optical diagnostic imaging comprises a novel group of imaging modalities that provide information by assessing differences between incident and detected light caused by the interaction of light with tissue. Scattering and absorption are tissue-specific optical properties and, by assessing these interactions,
diffeent tissue types can be distinguished.

Optical imaging has shown potential in several medical fields where they are employed routinely in various forms, ranging from pulse oximeters to fundus cameras, and experimental reports show promising results in the field of oncology [9].

Optical coherence tomography (OCT) is a technology developed in the early 1990s for ophthalmological applications [10] and is routinely used in that setting in current clinical practice. OCT is the optical equivalent of ultrasound, using light instead of sound to produce micrometer-scale resolution, cross-sectional images up to a depth of about 2 mm in renal tissue [Figure 2]. Resolutions up to 5 µm can be achieved, being 100–250 times higher than high-resolution ultrasound [11] and approaching that of microscopy. An image produced by OCT resembles the tissue structures observed in histology and can, therefore, be considered as an ‘optical biopsy’ [12] [Figure 2]. Moreover, data extracted from the original OCT images can be used for functional quantitative analysis after careful calibration of the OCT system. This finally results in a ‘functional optical biopsy’. The imaging depth is primarily limited by the scattering of light by cellular structures, hindering the return of reflections to the receiver. This scattering causes the light intensity to attenuate as it penetrates deeper into the tissue and this attenuation of OCT signal can be quantified by measuring the decay of signal intensity per unit depth. Using Lambert–Beer’s law and after careful calibration of the OCT system, a tissue specific attenuation coefficient (μOCT mm-1) can be derived [13–15]. Because malignant tissue displays an increased number, larger and more irregularly shaped nuclei with a higher refractive index and more active mitochondria, the μOCT is expected to be higher compared to normal and benign tissue [Figure 3].

In urology, the early research on OCT has been focused on tissue diagnosis predominantly in bladder and prostate cancer [12, 16] and, more recently, attention has turned to the field of renal cell carcinoma (RCC) and research is currently ongoing [17–20]. We were the first authors to publish data on the ability of OCT to differentiate renal malignancies from normal renal tissue using quantitative analysis. Subsequently, we performed an in vivo pilot study assessing the difference of the attenuation-coefficient of malignant renal tumours from normal renal parenchyma and benign tumours [18]. OCT-imaging took place using an in vivo OCT-probe during surgery, and a significant difference was found between the attenuation-coefficient of normal renal tissue and that of malignant tumours. Attenuation-coefficients of malignant and benign tumours did differ, although it is likely that the small sample size (3 benign tumours vs 11 malignant) is hindering a statistical significance, suggesting that a clear difference might be found in larger samples. Linehan et al. assessed qualitative differences of OCT images of different types of renal tumours showing that certain tumour subtypes do have different appearances on OCT-imaging; however, intriguingly, clinical distinction of tumours such as RCC from oncocytomas could not be demonstrated [19].

Future developments
Finally, anticipating the validation of results showing optical diagnostics being able to differentiate renal tissues, there is a potential role for the techniques in several clinical scenarios. Before going as far as replacing pathological examination as discussed earlier, the two techniques might be complementary with the real-time- and non-invasive nature of the optical techniques serving as guidance for correct needle placement in order to reduce the number of non-diagnostic biopsy results, as is already done in other malignancies, and the small in vivo probes necessary for such interventions are becoming commercially available. The technological configuration behind OCT allows for easy integration with diffuse reflectance spectroscopy (DRS) and Raman spectroscopy (RS). Moreover, the structural-imaging- and light-scattering based quantitative possibilities of OCT together with the quantitative light absorption sensitivity of DRS and the inelastic light scattering (and therefore biochemical) sensitivity of RS yields the full potential of a functional optical biopsy.

We would like to thank the Cure for Cancer Foundation (CFC) and the Technology Foundation (STW) for project funding. This work is part of the innovative Medical Imaging Technologies program (iMIT) of STW and the Novel Biopsy Methods program of CFC.

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The authors
D. Martijn de Bruin^1,2,* Msc; Peter G. Wagstaff¹ MD; Kurdo Barwari¹ PhD, MD; Ton G. van Leeuwen² PhD; Dirk J. Faber² PhD; Jean J. de la Rosette¹ PhD, MD; M. Pilar Laguna¹ PhD, MD.

*Corresponding author
E-mail: d.m.debruin@amc.uva.nl