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Featured Articles
A new biomarker for prostate cancer: [-2]proPSA
Prostate specific antigen (PSA) has significantly improved the early detection of prostate cancer (PCa), reducing the related mortality rate. However, PSA has a low specificity, being affected by many benign conditions. [-2]proPSA, a PSA precursor, is a more specific and accurate biomarker indicating prostate biopsy in men at real risk of PCa.
by Dr A. Abrate, Dr M. Lazzeri and Prof. G. Guazzoni
PSA as a marker for prostate cancer
Prostate specific antigen (PSA) is a serum marker widely used for the early detection of prostate cancer (PCa). Its introduction into clinical practice in the early 1990s had an extraordinary impact on the diagnosis and management of PCa. In fact, 20 years after its introduction, the PSA-based PCa opportunistic or systematic screening has resulted in a stage migration to more organ-confined tumours at the time of diagnosis, and consequently to a consistent reduction in PCa related mortality [1, 2]. However, PSA is not a perfect marker for the detection of PCa because of its low specificity and sensitivity. Its levels may increase as a result of benign conditions, such as benign prostatic hyperplasia (BPH) and chronic prostatitis. Moreover, PSA levels are also affected by biologic variability, which may be related to differences in androgen levels, prostate manipulation or ejaculation. Finally, alterations in PSA levels may be related to sample handling, laboratory processing, or assay standardization. All these factors made it difficult to find an appropriate PSA cut-off point diagnostic for PCa (for many years considered to be 4 ng/ml).
Thus, prostate biopsy is still mandatory to confirm the diagnosis. However, this is positive in only approximately 30% of patients [3], and the European Association of Urology suggests a repeat biopsy if PSA is persistently elevated, the digital rectal examination (DRE) is suspicious, or there is a pathological diagnosis of atypical small acinar proliferation (ASAP) or high-grade prostatic intraepithelial neoplasia (HG-PIN) [4]. Finally, PCa (also high-grade cancer) is not rare (approximately 15.2%) among men with PSA levels lower than 4 ng/ml, the previously widely accepted cut-off point [5].
Considering all these observations, it is clear that PSA is an organ-specific rather than an ideal cancer-specific marker.
The introduction into clinical practice of measuring the levels of several derivatives of PSA (free PSA, percentage of free PSA, PSA density, PSA velocity) improved the accuracy of total PSA (tPSA) in detecting PCa. Recently, free PSA (fPSA) has been found to include several subforms, such as proPSA. In particular [-2]proPSA seems to be specific for PCa, opening new ways for early cancer detection.
Biological basis of proPSA
The currently measurable serum tPSA consists of either a complexed form (cPSA, 70–90%), bound by protease inhibitors (primarily alpha1-antichymotrypsin), and a non-complexed form (fPSA). Recently fPSA has been discovered to exist in at least three molecular forms: proPSA, benign PSA (BPSA), and inactive intact PSA (iPSA), covering approximately 33%, 28%, and 39% of fPSA respectively (Fig. 1) [6]. In particular, proPSA is a proenzyme (precursor) of PSA, which is associated with PCa [7].
PSA is synthesized with a 17-amino acid leader sequence (preproPSA) that is cleaved co-translationally to generate an inactive 244-amino acid precursor protein (proPSA, with seven additional amino acids compared to mature PSA). proPSA is normally secreted from the prostate luminal epithelial cells. Immediately after its release into the lumen, the pro-leader part is removed, creating the active form, by the effect of human kallikrein (hK)-2 and hK-4, which have a trypsin-like activity and are expressed predominantly by prostate secretory epithelium. Other kallikreins, localized in the prostate, such as hK216 or prostin17, are involved in the conversion and activation of proPSA. Cleavage of the N-terminal seven amino acids from proPSA generates the active enzyme, which has a mass of 33 kDa.
The partial removal of this leader sequence leads to other truncated forms of proPSA. Thus, theoretically seven isoforms of proPSA could exist, although only [-1], [-2], [-4], [-5], [-7]proPSA were found; there is still no evidence of [-3], [-6]proPSA. However, all forms of proPSA are enzymatically inactive [8]. It is possible to detect three truncated forms of proPSA in serum: [-5/-7], [-4] and [-2]proPSA, which is the most stable form (Fig. 1).
Notably, in vitro experiments showed that the [-2]proPSA form cannot be activated by either hK2 or trypsin; thus, once it is formed, [-2]proPSA is resistant to activation into the mature PSA form and consequently this is the most reliable test.
Mikolajczyk et al. [7], using a monoclonal antibody recognizing [-2]proPSA, found increased staining in the secretions from malignant prostate glands. In particular [-2]proPSA is differentially elevated in peripheral gland cancer tissue; conversely transition zone tissue contains little or no proPSA.
The increased serum tPSA concentrations in patients with PCa do not result from increased expression but rather from an increased release of PSA into the bloodstream, due to disruption of the epithelial architecture. fPSA is catalytically inactive because of internal cleavages, occurring in seminal plasma, and does not form complexes with protease inhibitors or other proteins: in PCa %fPSA is lower presumably because, consequently to an increased release of PSA into the bloodstream, a very low part is still degraded into the ducts.
In another later study [9], Mikolajczyk et al. found that [-2]proPSA was specifically higher in patients with PCa. Analysing a small number of patients with biopsy positive for PCa and tPSA between 6 and 24 ng/ml, they found that [-2]proPSA constituted a high fraction of fPSA (25% to 95%), which was greater than in patients with a negative biopsy. However, the molecular basis for the proPSA elevation in PCa is uncertain, although a decreased cleavage by hK2 could be the cause.
Clinical utility of proPSA
Sokoll et al. [10] were the first to study the role of proPSA in the early detection of PCa. The study involved archival serum from 119 men (31 PCa, 88 non-cancer), obtained before biopsy and in the tPSA range of 2.5–4.0 ng/ml. The serum levels of tPSA, fPSA, proPSA, and proPSA/fPSA ratio (%proPSA) were analysed: PSA and %fPSA values were similar between the non-cancer and PCa groups, and %proPSA was relatively higher in the PCa group (50.1±4.4%) compared to the non-cancer group (35.5±6.7%; P=0.07). Concerning the clinical utility, the area under the curve (AUC) for %proPSA was 0.688 compared to 0.567 for %fPSA. At fixed sensitivity of 75%, the specificity was significantly greater for %proPSA at 59% compared with %fPSA at 33% (P<0.0001).
Afterwards, the Prostate Health Index (PHI) has been proposed as a mathematical algorithm combining tPSA, fPSA and [-2]proPSA according to the formula: ([-2]proPSA/fPSA) × √tPSA.
A large American prospective trial [11], involving 892 men who had tPSA levels of 2–10 ng/ml and negative digital rectal examination results, showed that PHI had greater predictive accuracy for prostate biopsy outcome (AUC 0.703) than [-2]proPSA (AUC 0.557), %fPSA (AUC 0.648) and PSA (AUC 0.525), directly correlating with Gleason score (GS) (P=0.013), with an AUC of 0.724 for GS ≥4+3 disease. Moreover, men with PHI >55 had a 42% likelihood of being diagnosed with high-grade disease on biopsy compared to 26% of men with PHI 0–24.9.
Accordingly, an observational European multicenter cohort study involved 646 men with tPSA levels of 2–10 ng/ml, who had undergone prostate biopsy [12]. [-2]proPSA and PHI improved the predictive accuracy for the detection of overall PCa (and also GS ≥7 disease) compared to PSA and derivatives. In fact, at 90% sensitivity, the PHI cut-off of 27.6 could avoid 100 (15.5%) biopsies, missing 26 (9.8%) cancers (23 with GS 6, three with GS 3+4).
Moreover, a PHI based nomogram to predict PCa at extended prostate biopsy was developed and validated over 729 patients [13]. Including PHI in a multivariable logistic regression model, based on patient age, prostate volume, digital rectal examination and biopsy history, significantly increased predictive accuracy by 7% from 0.73 to 0.80 (P<0.001). Decision curve analysis showed that using the PHI based nomogram resulted in the highest net benefit.
Recently, it was demonstrated that PHI might have a role in screening patients at high risk of PCa [14]. Specifically, the study involved 158 men with a positive family history undergoing prostate biopsy within the multicentre European PROMEtheuS cohort. Similarly to previous studies in the general population, PHI outperformed tPSA and %fPSA for PCa detection on biopsy (AUC 0.73, 0.55 and 0.60, respectively). In addition, both [-2]proPSA and PHI were directly associated with GS in men with a positive family history. Overall, the authors reported that using a PHI cutoff value of 25.5 would have avoided 17.2% of biopsies while missing only two GS 7 cancers. On decision curve analysis, the addition of PHI to a base predictive model that included age, prostate volume, tPSA, fPSA and %fPSA resulted in net benefit at threshold probabilities of 35–65%. This result suggests that PHI should be incorporated into a multivariable risk assessment for high-risk patients because it offers improved performance for PCa detection.
Conclusions
[-2]proPSA and PHI are more accurate than the currently used tests (PSA and derivatives) in predicting the presence of PCa at biopsy. Their implementation in clinical practice has the potential to significantly increase physicians’ ability to detect PCa and avoid unnecessary biopsies. Further work is needed to confirm and generalize these data on wider populations.
References
1. Hoffman RM, Stone SN, Espey D, Potosky AL. Differences between men with screening-detected versus clinically diagnosed prostate cancers in the USA. BMC Cancer 2005; 5: 27.
2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013; 63: 11–30
3. Vickers AJ, Cronin AM, Roobol MJ, et al. The relationship between prostate-specific antigen and prostate cancer risk: the Prostate Biopsy Collaborative Group. Clin Cancer Res. 2010; 16: 4374–4381.
4. Heidenreich A, Bellmunt J, Bolla M, et al. EAU guidelines on prostate cancer. Part 1: screening, diagnosis, and treatment of clinically localised disease. Eur Urol. 2011; 59: 61–71.
5. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med. 2004; 350: 2239–2246.
6. Mikolajczyk SD, Rittenhouse HG. Pro PSA: a more cancer specific form of prostate specific antigen for the early detection of prostate cancer. Keio J Med. 2003; 52: 86–91.
7. Mikolajczyk SD, Millar LS, Wang TJ, et al. A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue. Cancer Res. 2000; 60: 756–759.
8. Jansen FH, Roobol M, Jenster G, Schroder FH, Bangma CH. Screening for prostate cancer in 2008 II: the importance of molecular subforms of prostate-specific antigen and tissue kallikreins. Eur Urol. 2009; 55: 563–74.
9. Mikolajczyk SD, Marker KM, Millar LS, et al. A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer. Cancer Res. 2001; 61: 6958–6963
10. Sokoll LJ, Chan DW, Mikolajczyk SD, et al. Proenzyme psa for the early detection of prostate cancer in the 2.5–4.0 ng/ml total psa range: preliminary analysis. Urology 2003; 61: 274–276.
11. Catalona WJ, Partin AW, Sanda MG, et al. A multicenter study of [-2]pro-prostate specific antigen combined with prostate specific antigen and free prostate specific antigen for prostate cancer detection in the 2.0 to 10.0 ng/ml prostate specific antigen range. J Urol. 2011; 185: 1650–1655.
12. Lazzeri M, Haese A, de la Taille A, et al. Serum isoform [-2]proPSA derivatives significantly improve prediction of prostate cancer at initial biopsy in a total PSA range of 2-10 ng/ml: a multicentric European study. Eur Urol. 2013; 63: 986–994.
13. Lughezzani G, Lazzeri M, Larcher A, et al. Development and internal validation of a Prostate Health Index based nomogram for predicting prostate cancer at extended biopsy. J Urol. 2012; 188: 1144–1150.
14. Lazzeri M, Haese A, Abrate A, et al. Clinical performance of serum prostate-specific antigen isoform [-2]proPSA (p2PSA) and its derivatives, %p2PSA and the prostate health index (PHI), in men with a family history of prostate cancer: results from a multicentre European study, the PROMEtheuS project. BJU Int. 2013; 112: 313–321.
The author
Alberto Abrate* MD; Massimo Lazzeri MD, PhD; and Giorgio Guazzoni MD
Dept of Urology, Ospedale San Raffaele Turro, San Raffaele
Scientific Institute, Milan, Italy
*Corresponding author
E-mail: alberto.abrate@gmail.com
The relevance of the manufacturer in indirect immunofluorescence standardization
Autoantibody detection is a powerful laboratory tool for clinical diagnosis in the autoimmune diseases field. Among the techniques most widely used worldwide, indirect immunfluorescence (IFA) plays a particularly important role not only in the diagnosis but in the follow up of many diseases and remains the hallmark despite the introduction of new techniques in the routine of clinical laboratories. Witness to this is the renaissance of the antinuclear antibodies (ANA) screening on HEp2 cells by this techique or the renewal of the detection of anti-endomysium antibodies on monkey esophagus as the gold standard serological test for celiac disease. Therefore, IFA is a technique in full validity and requires a level of standardization that unfortunately is far from being achieved.
by Petraki Munujos, PhD
The efforts to improve standardization of indirect immunofluorescence as a diagnostic tool are numerous worldwide. Traditionally, the players involved in standardization have been clinical laboratories, clinicians, regulators, and to a lesser degree, diagnostic reagents manufacturers. Energy has been concentrated basically in aspects like the control of laboratory procedures, unification of nomenclatures and classifications, guidelines on how to report the results, preparation of recommendations, definition of diagnostic criteria and diagnostic algorithms and development of external quality control programs. In these iniatives, laboratory staff, clinicians and regulators are mainly involved. Nevertheless, those aspects regarding the design, development and manufacturing of the reagents, which involve manufacturers, are basically ignored. And this is probably due to the fact that the evolution of the technology has led to a truncated view of the test procedure resulting in a misconception of what needs to be standardized. In other words, the execution of many procedures is nowadays being shared between the manufacturer, who actually initiates the assay, and the laboratory, where the test is finalized. In old scientific articles related to ANA, the Material and Methods section usually started with the cell culture, the preparation of the slides and the fixation among others, and the sample incubation was only one more step of the whole procedure. Currently, the Material and Methods section starts with the sample preparation and instead of describing all the preliminary steps, one can find the name and references of the manufacturer. Figure 1 illustrates what would be the whole test procedure, showing the part performed in the clinical laboratory, actually the only part which is taken into consideration when dealing with standardization. So, to ensure appropriate use of indirect immunofluorescence testing, clinicians, diagnostic laboratories, regulators and reagents manufacturers should be involved and share the tasks of identifying and managing the key points leading to proper results.
Evidences of disparity
At the level of the manufacturer, the potential variability in the performance of the kits lies in features like the reagents and materials that are purchased or manufactured to become components of the kit, the procedures and conditions of manufacturing (fixatives, temperatures, formulations), the reliability of the serum samples used to set up the calibration of the determination (basically, the sample dilution which actuallly acts as the cut-off point), and the stability of the final product (1).
When approaching the participation of the manufacturer in the standardization of antibody testing, it is observed that what basically matters for industry is the standardization of the manufacturing processes. This normally occurs in an environment of Quality System Certifications, like GMP, ISO-9001 or ISO-13485 and under the requirements of the European Directive on In Vitro Medical Devices, and it is strengthened by the manufacturer’s own interest in having robust and reliable processes. Nevertheless, despite regulatory compliant and well implemented standardized processes, there are several aspects that make final reagents differ from one manufacturer to another. Below are reviewed some examples of variation on the results depending on the manufacturer source.
Dense fine speckles 70 (DFS70) antigen
As with other fluorescence patterns, the typical DFS pattern (lens epithelium-derived growth factor) can vary depending on the manufacturer source of the HEp2 slides used. The variations consist basically in different sensitivities and even in positive and negative results for the same sample run in different slide brands. Inconsistencies are also observed when comparing fluorescence with the results obtained by means of ELISA (2,3).
Ribosomal P protein (Rib P)
In studies performed by Mahler et al. (4) to determine the sentitivity of the immunofluorescence technique to detect antibodies against ribosomal P protein, several different HEp2 slides manufacturers were used, resulting in significant differences in patterns of staining for monospecific anti-Rib-P sera. Differing patterns were observed for the same sample, from a fine speckled nucleoplasmic pattern, to a diffuse cytoplasmic staining, or a fine speckled cytoplasmic pattern.
CDC/AF Reference Human Sera
When running reference sera on HEp2 slides coming from different manufacturers, variations of unknown origin can be observed. While most brands produce the expected specific pattern, there are often differences among brands like the ones shown in Figure 2.
Labile nuclear antigens
Most of the patterns observed when analysing the presence of ANA in patients sera by IFA on HEp2 cells slides are suitably detected in most slides brands. However, there are some antigens for which expression may significantly vary from one manufacturer to another like Jo1, PCNA or SSA/Ro (5). These antigens are not always well preserved in the substrates and they can be extremely sensitive to handling, to certain fixatives and in some cases, they can be just washed out during the manufacturing process, resulting in a poor presence or a total lack of antigenic molecules available to capture the antibody being analysed.
Antineutrophil cytoplasmic antibodies (ANCA)
The neutrophil substrates used in the detection of ANCA may vary in their ability to give the typical immunofluorescence patterns described and established by consensus groups, i.e. a diffuse granular cytoplasmic staining with higher interlobular intensity (C-ANCA), a compact staining of the perinuclear zone of the cytoplasm (P-ANCA) and a broad non homogeneous perinuclear staining, eventually accompanied by a diffuse cytoplasmic pattern with no accentuation of the interlobular zone (X-ANCA). In general, substrates differ in their ability to distinguish between a C-ANCA and X-ANCA. In a study by Pollock et al. (6), it was observed that although all commercial neutrophil substrates consistently demonstrated nuclear extension of perinuclear fluorescence with sera containing P-ANCA with MPO specificity, there were more problems in P-ANCA testing than in C-ANCA, due basically to the eventual presence of additional cytoplasmic fluorescence.
Crithidia luciliae
In a similar way as observed in HEp2 cells immunofluorescence patterns, the anti-nDNA test on Crithidia luciliae slides may show significant differences among manufacturers. The variety of strains available in cell banks contribute to the heterogeneity of results. Apart from the kinetoplast, other organelles can be stained by antibodies from the sample, like the nucleus, the basal body and the flagellum. Depending on the conditions of preparation of C. luciliae substrates and on the nature of the sample analysed, different patterns of stained organelles can be observed. Nevertheless, the only specific staining to be considered as a positive result is the kinetoplast staining. In addition to anti-nDNA antibodies, there are other antibodies in the serum of lupus patients that can react with the substrate. The so called anti-nucleosome antibodies are antibodies that react with histones exposed in the nucleosome. It is well known that treating C. luciliae substrate with HCL eliminates histone from the kinetoplast (7). This could be another point of possible discrepancy among manufacturing processes if some include the histone removal procedure and some others do not. Furthermore, the cell cycle of C. luciliae may influence histone appearance in the kinetoplast. Therefore, the manufacturing process of C. luciliae slides, including culture, harvest, fixation and drying, can cause variation in the results.
Aspects providing variablity
Among the players participating in autoimmune diagnostics, there is no doubt that manufacturers hold the know-how of preparing diagnostic kits and are the true experts in the development of test methods. However, despite the standardized manufacturing processes and the CE-certifications or FDA approvals, there are several aspects that are found to be sources of variabilty. These aspects should be addressed and recommendations on key points should be created by specialized committees with the participation of laboratory experts, clinicians and manufacturers. The definition and control of the raw materials incorporated in the kit production is a common and regulated practice in any kind of manufacturing process. But recommendations on nature, compostion or quality grades of key materials, including culture media, cell type and strain or fluorescent conjugates is still lacking. In the case of tests based on cellular substrates, extracellular matrix (ECM) proteins are commonly used to aid the spreading and growth of cells on the slide glass surface. Many ECM proteins contain defined amino acid sequences to which cell surface integrin receptors bind specifically. ECM, together with growth factors in the culture medium, work to produce an appropriate in vitro proliferative response, promoting cell growth and spreading. Altering cell-ECM contacts results in coordinated changes in cell, cytoskeletal, and nuclear form. Thus, the choice of the right ECM to coat the glass slides used as growing surface deserves our attention since it might have a direct effect on the fluorescent pattern finally observed (8). It is also common to use synchronization agents to achieve a greater rate of mitotic cells. Due to the fact that these compounds may be toxic for the cell, some cell disturbances may occur that can impact the morphology or the behaviour of the final cell preparation.
Diagnosis by means of tissue sections remains very important in liver autoimmune diseases like autoimmune hepatitis (AIH) or primary billiary cirrhosis (PBC). In particular, the detection of anti-smooth muscle antibodies (ASMA), antibodies to liver-kidney microsomes (LKM antibodies) and anti-mitochondrial antibodies (AMA) are considered important diagnostic tools. Only a few guidelines have been published on the obtention of tissue sections (9), while the variations in the preparation of tissue blocks regarding orientation, preservation conditions, and sectioning keep on contributing to the heterogeneity of results, especially in the case of tissues that are not morphologically homogeneous. For instance, the LKM antibodies can only be well defined if the kidney section has the proper orientation that allows the distinction between proximal and distal renal tubules and, thus, between LKM and AMA.
Considering that the expression and topographical distribution of autoantigens is under the direct influence of the HEp-2 fixation method, some immunofluorescence patterns are not adequately expressed due to the way that the antigenic substrate is prepared. This aspect equally affects tissue and cell substrates. As for the sensitivity of the tests, differences among manufacturers are due to the use of fixatives to prolong shelf-life. The use of slides without fixation seems to be the best choice for most autoantibody patterns. Nevertheless, there are several staining patterns that need the substrate to be fixed (figure 3), like anti-islet cells antibodies or anti-adrenal cortex antibodies.
A less frequent but significant source of variability in the immunofluorescence on tissue sections can be found in the origin of the animal used (Figure 4). Definition of suitable species and strains should be addressed in some cases in which the levels of antigen expression may differ. This affects the sensitivity of the test, especially in samples with moderate or low titers of antibody.
Considering the complexity and diversity of manufacturing processes and subprocesses and their impact on the final test performance, it is important to combine the efforts of laboratory experts, clinicians and manufacturers in the task of standardizing those key aspects that could otherwise keep on undermining the successful harmonization of the results obtained in the clinical laboratory.
References
1. Fritzler MJ, Wiik A, Fritzler ML, Barr SG. The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 2003, 5:192-201
2. N.Bizzaro, E.Tonuttiand D.Villalta, «Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al,» Arthritis Rheum, vol. 63, no. 12, pp. 4036-4037, 2011
3. Mahler M. The clinical significance of anti-DFS70 antibodies as part of ANA testing. In: K. Conrad, E.K.L. Chan, M.J. Fritzler, R.L. Humbel, P.L. Meroni, G. Steiner, Y. Shoenfeld (Eds.). Infection, Tumors and Autoimmunity, AUTOANTIGENS, AUTOANTIBODIES, AUTOIMMUNITY, Volume 9, p.342-350. PABST, 2013.
4. Mahler M, Ngo JT, Schulte-Pelkum J, Luettich T, Fritzler MJ. Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies. Arthritis Research & Therapy 2008, 10:R131
5. Dellavance A, de Melo Cruvinel W, Carvalho Francescantonio PL, Pitangueira Mangueira CL, Drugowick IC, RodriguesSE; Coelho Andrade LE. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides J Bras Patol Med Lab 2013;49( 3):182-190.
6. Pollock W, Clarke K, Gallagher K, Hall J, Luckhurst E, McEvoy R, Melny J, Neil J, Nikoloutsopoulos A, Thompson T, Trevisin M, Savige J. Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate. J Clin Pathol 2002;55:680–683
7. Kobkitjaroen J, Jaiyen J, Kongkriengdach S, Potprasart S, Viriyataveekul R. Comparison of Three Commercial Crithidia luciliae Immunofluorescence Test (CLIFT) Kits for Anti-dsDNA Detection. Siriraj Med J 2013;65:9-11
8. (Integrin Binding and Cell Spreading on Extracellular Matrix Act at Different Points in the Cell Cycle to Promote Hepatocyte Growth Hansen LK,. Mooney DJ, Vacanti JP, Ingber DE. Molecular Biology of the Cell 1994;5:967-975
9. Vergani D, Alvarez F, Bianchi FB, Cançado ELR, Mackay IR, Manns MP, Nishioka M, Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. Journal of Hepatology 2004;41: 677–683
Progress in biomarkers of colorectal cancer
Colorectal cancer is one of the commonest types of cancer and contributes significantly to cancer-related mortality. Recent research has focused on the identification, development, validation of sensitive and specific biomarkers to improve early diagnosis, to assess disease outcome, accurately predict response to therapy or monitor disease status after treatment.
by Dr Caroline Coghlin and Professor Graeme I Murray
The requirement for biomarkers in colorectal cancer
Colorectal cancer (CRC) represents a heterogeneous disease with variable clinical presentations and equally variable outcomes observed between individual patients. At the molecular level, although several well-described pathways are known to exist in CRC tumorigenesis the reality is likely to be much more complex. In addition to genetic alterations, numerous interactions exist between multiple abnormal signalling networks and between the tumour cells and their microenvironment. Surgery is the mainstay of treatment in primary CRC but options for adjuvant or neoadjuvant chemotherapy are advancing. Given these choices, and the potential toxicity associated with some agents used, the need for validated and reliable biomarkers to aid in CRC diagnosis, management and predictive and prognostic stratification is growing [1].
Diagnostic, prognostic and predictive biomarkers
The detection of protein biomarkers using immunohistochemistry in fixed tissue is a reliable technique widely used in the histopathology laboratory setting. Accurate diagnosis of primary CRC is often clear from its site of origin detected colonoscopically or with imaging modalities and from the morphological features revealed on histology. However, diagnosis of CRC in secondary sites can be more difficult. CRC cells frequently express CK20 and CDX2 (an intestinal-specific transcription factor) and they are generally negative for CK7. Therefore, a targeted panel of immunohistochemical markers can aid in the diagnosis of metastatic CRC and so guide appropriate treatment [2].
Accurate prognosis in CRC currently relies on pathological and clinical staging including use of the tumour, node, metastasis (TNM) system. In the past, assay of plasma biomarkers such as carcinoembryonic antigen (CEA) and CA19.9 to aid in prognostic stratification was advocated but, while these markers may still have a role in disease monitoring after surgery, as diagnostic and prognostic tools such biomarkers lack sensitivity and specificity. A large body of research has focused on comparative proteomic analysis of primary site CRC, normal control tissue and metastatic tissue in attempts to identify clinically useful prognostic and predictive CRC biomarkers [3, 4]. Candidate markers have included the mismatch repair proteins, especially MLH1 and MSH2 (which may be linked with a better prognosis in early CRC but also with a poor response to some chemotherapeutic agents such, as 5-fluorouracil) along with many other molecules involved in diverse cellular processes such as matrix degradation, oxidative metabolism or protein folding [3, 4]. There is, however, often a deficiency in the consistent follow-up studies necessary for the validation of such potential biomarkers. This aspect of study remains crucial to enable the robust and reliable clinical application of biomarkers in CRC [Fig. 1].
Plasma/serum biomarkers in CRC
The goal of screening programmes for CRC is to decrease mortality and reduce morbidity associated with the disease by identifying cancers at an early stage when intervention is more likely to succeed. Fecal occult blood or DNA testing and targeted colonoscopy are methods commonly employed to screen for early lesions. Fecal testing has the disadvantage of poor patient acceptability and it also lacks sensitivity. Colonoscopy, while accurate, is invasive and associated with a small but significant risk to the patient. With this in mind, the ultimate aim of many recent studies has been to identify robust plasma-based biomarkers which represent an acceptable form of patient testing. Such biomarkers should ideally be able to identify early invasive or perhaps even pre-invasive CRC in a sensitive and specific manner.
Circulating tumour cells (CTCs) are malignant cells originating from a primary tumour or its metastases which have gained access to the bloodstream. Several methods have recently been developed to identify these cells which may be associated with a poorer prognosis or an increased risk of relapse after surgery. In addition, molecular genetic characterisation of CTCs has potential implications for targeted therapy and therefore predictive value. CTCs in CRC patients are more abundant in central blood compartments such as the mesenteric vessels but peripheral blood can also be a source providing enrichment techniques are employed. In patients with metastatic CRC identification of CTCs in peripheral blood is associated with an adverse prognosis [5]. In addition, post-operative measurement of CTC levels may be used to predict tumour recurrence after surgery [6]. However, attempts to link CTC levels in early stage CRC (stage 1 disease) with an adverse prognosis have shown less convincing results and therefore the utility of this method as a screening modality is currently unclear [7].
Increased levels of circulating cell-free DNA (cfDNA) in cancer patients have been studied as a potential biomarker of the disease [8]. The precise source of these cfDNA fragments in conditions such as CRC remains controversial but tumour cell apoptosis, necrosis, or possibly active secretion have been suggested as putative mechanisms. A recent study used quantitative real-time PCR to detect ALU repeats in the plasma or serum of operated or non-operated CRC patients and compared the levels of circulating cfDNA detected with normal healthy controls [9]. The results showed that circulating levels of cfDNA were significantly higher in non-operated CRC patients when compared with operated patients and controls. This study concluded that quantification of serum cfDNA could be important in detecting and monitoring CRC patients in both early and late stage disease. The predictive value of cfDNA analysis has also been suggested. A subset of patients with initial wild type KRAS status receiving monotherapy with epidermal growth factor receptor (EGFR) inhibitors have been shown to develop early KRAS mutations detectable in cfDNA in the serum months before imaging revealed disease progression [10]. Despite progress in the detection and monitoring of cfDNA there are no reproducible studies to date to show direct and consistent correlation between CRC stage and circulating cfDNA levels. As laboratories use different methods to detect, analyse and monitor cfDNA, future translation to clinical use will require further standardization to ensure consistency in analysis.
MicroRNAs (miRNAs) are small non-coding RNA strands that can post-transcriptionally regulate the expression of multiple target genes. They have been implicated in several steps in carcinogenesis and their measurement in the serum of CRC patients has shown early promise for disease detection and monitoring of cancer recurrence after surgery [8]. miRNA-29c is thought to suppress tumorigenesis by inhibiting cell proliferation and migration. Circulating miRNA-29c levels have been studied as a potential biomarker for both early and late recurrence following surgery in colorectal cancer [11]. The oncogenic miRNA, miRNA-21, which negatively regulates tumour suppressor genes, has shown promise as a possible diagnostic and prognostic marker in CRC. Levels of this miRNA were significantly raised in CRC patients and patients with colonic adenomas when compared with healthy controls [12]. Serum miRNA-21 levels also fell in those patients undergoing curative surgery. Increased levels of this miRNA in both tumour tissue and the patients’ serum were found to be significantly associated with tumour size, the presence of metastasis and reduced survival. A possible drawback of miRNA analysis in cancer patients is the variable extraction rates of these small molecules from patients’ plasma or serum. To date this has produced inconsistent results between different studies. Once again a standardized approach will be required.
Proteomic analysis of tissue samples in CRC has been proven a valuable technique for the identification of potential biomarkers. Given the issues surrounding patient acceptability with the use of faecal material or invasive techniques in CRC screening, recent studies have focused on identifying plasma or serum protein biomarkers which can aid in the early diagnosis of CRC. Choi et al. analysed plasma samples from patients with colorectal adenomas or invasive disease and identified a panel of proteins, including three cytokines, which were differentially expressed between the study groups [13]. Another group used combinations of serum CEA, cytokines and CA19.9 to try to differentiate adenoma-bearing patients from healthy controls or those with established CRC [14]. Multiplex protein arrays have been developed to analyse serum samples from CRC patients, adenoma-bearing patients and healthy controls. Initial results indicated that combinations of CEA and IL-8 or CEA and C-reactive protein showed the best screening performance for early CRC or adenoma detection [15]. However, although the overall specificity of the tests employed was relatively high, the sensitivity was much lower (particularly with regard to adenoma detection) and the authors concluded that clinical use of such novel systems could be made in combination with established techniques such as faecal testing, for screening purposes.
Conclusions
Although significant progress has been made recently in the development of biomarkers in CRC there is still a relative lack of consistent follow-up data available for the validation of such markers. This aspect of research needs to be addressed in order to facilitate the transition of putative biomarkers from the research stage into robust and reliable clinical applications. The search for acceptable plasma-based biomarkers to aid in screening is gaining momentum but perhaps in future a combination of techniques will be required to accurately guide early diagnosis and intervention in colorectal cancer.
References
1. De Wit M, Fijneman RJ, Verheul HM, et al. Proteomics in colorectal cancer translational research: biomarker discovery for clinical applications. Clin Biochem. 2013; 46: 466–479.
2. Coghlin C, Murray GI. Following the protein biomarker trail to colorectal cancer. Colorectal Cancer 2012; 1: 93–96.
3. Ralton LD, Murray GI. Biomarkers for colorectal cancer: identification through proteomics Curr Proteomics 2010; 7: 212–221.
4. O’Dwyer D, Ralton LD, O’Shea A, Murray GI. The proteomics of colorectal cancer: identification of a protein signature associated with prognosis. PLoS One 2011; 6: e27718.
5. Groot Koerkamp B, Rahbari NN, Büchler MW, Koch M, Weitz J. Circulating tumor cells and prognosis of patients with resectable colorectal liver metastases or widespread metastatic molorectal mancer: a meta-analysis. Ann Surg Oncol. 2013; 20: 2156–2165.
6. Galizia G, Gemei M, Orditura M, et al. Postoperative detection of circulating tumor cells predicts tumor recurrence in colorectal cancer patients. J Gastointest Surg. 2013; Epub ahead of print, doi: 10.1007/s11605-013-2258-6.
7. Linuma H, Watanabe T, Mimori K et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes’ stage B and C colorectal cancer. J Clin Oncol. 2011; 29: 1547–1555.
8. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011; 11: 426–437.
9. da Silva Filho BF, Gurgel AP, Neto MÁ, et al. Circulating cell-free DNA in serum as a biomarker of colorectal cancer. J Clin Pathol. 2013; 66: 775–778.
10. Misale S, Yaeger R, Hobor S et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486: 532–536.
11. Yang I-P, Tsai H-L, Huang C-W, et al. The functional significance of microRNA-29c in patients with colorectal cancer: A potential circulating biomarker for predicting early relapse. PLoS One 2013; 8: e66842.
12. Toiyama Y, Takahashi M, Hur K, et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. J Natl Cancer Inst. 2013; 105: 849–859.
13. Choi JW, Liu H, Shin D H, et al. Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients. Proteomics 2013; 13: 2361–2374.
14. Pengjun Z, Xinyu W, Feng G, et al. Multiplexed cytokine profiling of serum for detection of colorectal cancer. Future Oncol. 2013; 9: 1017–1027.
15. Bünger S, Haug U, Kelly M, et al. A novel multiplex-protein array for serum diagnostics of colon cancer: a case-control study. BMC Cancer 2012; 12: 393. doi: 10.1186/1471-2407-12-393.
The authors
Caroline Coghlin1 BA, BM Bch, DRCOphth, FRCPath; Graeme I. Murray2* MB ChB, PhD, DSc, FRCPath
1Department of Pathology, Aberdeen Royal Infirmary, NHS Grampian, Aberdeen, UK
2Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK
*Corresponding author
E-mail: g.i.murray@abdn.ac.uk
NIPT: an opportunity for early detection and promising treatment for children with XXY
Inconsistent detection and false-positive rates have plagued traditional screening measures for trisomy, thus encouraging the development of less risky and invasive measures. Through the advent of single-nucleotide polymorphism-based and informatics-based non-invasive prenatal testing, accurate detection of trisomies 13, 18, 21 as well as the X and Y chromosomal aneuploidies of XXY, XYY and XXX in early in pregnancy is now possible. This technology is extremely important in ensuring infants with these disorders are identified in a timely manner so that proper care and treatment can be administered for optimal development.
by Emily J. Stapleton, Dr Megan Hall and Dr Carole A. Samango-Sprouse
Cell-free DNA-based non-invasive prenatal testing
Traditional serum- and ultrasound-based screens have high false-positive rates and less-than-ideal detection rates, resulting in unnecessary and risky invasive procedures and missed diagnoses [1]. The discovery of fetal cell-free DNA (cfDNA) in maternal circulation allowed the development of a more accurate, non-invasive approach for fetal aneuploidy screening [termed non-invasive prenatal testing (NIPT)] [2]. However, cfDNA is highly fragmented and is heavily diluted with maternal cfDNA [3]. Hence, methods to accurately detect fetal aneuploidies using cfDNA analysis had to overcome these technical limitations. Two approaches to-date have accomplished this and have been successfully commercialized. The first-generation quantitative ‘counting’ approaches amplify and sequence non-polymorphic loci and compare absolute quantities of DNA from the chromosome(s) of interest (e.g. chromosome 21) to that of reference chromosomes [4]. The second, next-generation approach specifically amplifies and sequences single-nucleotide polymorphisms (SNPs), identifying both allele identity and distribution [4].
First-generation quantitative counting methods
The most straight forward counting methods non-specifically amplify cfDNA, followed by massively parallel shotgun sequencing (MPSS) [4]. A more recent approach uses targeted amplification and sequencing, thus improving efficiency [4]. Both methods amplify non-polymorphic loci, and identify fetal aneuploidy by detecting abnormally high or low amounts of cfDNA from the chromosome(s) of interest relative to internal reference chromosomes that are presumably euploid in the fetus. If the proportion of reads associated with a particular chromosome relative to the reference chromosome(s) is found to be significantly above the expected proportion for a euploid fetus, the extra reads are presumed to have originated from an extra chromosome present in the fetal genome and fetal trisomy is inferred. Counting methods have shown remarkable improvements over serum screening and ultrasound methods, reporting >97% sensitivity for trisomies 21 and 18, and false positive rates of <0.2% for trisomy 21 [4]. However, the false positive rate can be as high as 1% for other indications [4]. Additionally, counting methods have reduced sensitivity when detecting aneuploidy of chromosomes 13 and X [4]. This is thought to be due to a combination of variable amplification efficiency due to decreased guanosine–cytosine content, as well as unusual biology specific to the X chromosome. Significantly, the requirement for a reference chromosome renders these methods unable to detect triploidy.
A next-generation approach for NIPT: analysing SNPs
The next-generation PanoramaTM test is the only commercialized NIPT that incorporates genotypic information, in the form of SNPs, to accurately identify fetal chromosomal copy number from cfDNA [5, 6]. This allows a more complex and nuanced cfDNA analysis than first-generation methods that do not take into account genotypic information and only consider the number of reads. This SNP-based approach is able to identify both the allele identity and distribution, thus identifying the maternal and fetal cfDNA contribution to the sequence reads. Additionally, Panorama uses a sophisticated bioinformatics algorithm called Next-generation Aneuploidy Testing Using SNPs (NATUS) that leverages advanced Bayesian statistics.
The NATUS algorithm incorporates parental genotypic information to aid analysis of relatively noisy measurements that result from the mixture of maternal and fetal cfDNA. Specifically, NATUS considers the maternal genotype, which is obtained by measuring genomic DNA isolated from white blood cells present in the maternal blood sample, as well as the paternal genotype, if available (though not necessary); the algorithm incorporates crossover frequency data from the human genome project to bioinformatically predict all of the possible fetal genotypes that could arise from the parental genotypes. These billions of hypotheses are then compared to the actual cfDNA measurements, and a likelihood is calculated for each hypothesis. The hypothesis with the maximum likelihood indicates the actual genetic state of the fetus, thus determining the presence or absence of a chromosomal abnormality.
This approach enables the incorporation of many more quality control metrics, improving accuracy over first-generation counting approaches. First, it creates the ability to flag samples with additional abnormalities, including samples with large deletions and duplications, mosaicism, and extra parental haplotypes, which indicate undetected twins, vanishing twins, or triploidy; any of these may result in miscalls with first-generation NIPTs. Second, the algorithm can take into account a number of other indicators of accuracy in addition to fetal fraction, for example the total amount of cfDNA in the sample, and the degree of contamination. This allows the algorithm to determine when the data is insufficiently clear to make an accurate call, even if the fetal fraction is above the minimum threshold of 3.8%; this reduces the number of incorrect calls. Third, this approach does not rely on a reference chromosome, which enables highly accurate detection of abnormalities on chromosomes that do not amplify with reliable efficiency, such as chromosome 13 and the sex chromosomes, as well as the unique ability to detect triploidy [5, 6]. These advantages, therefore, overcome limitations of the first-generation approach.
This translates to a quantifiable improvement in performance [6]. Specifically, in clinical studies, the NATUS algorithm showed 100% sensitivity when detecting trisomy 21, trisomy 18, trisomy 13, fetal sex, and triploidy, and of 91.7% when detecting monosomy X (Turner syndrome) [5, 6]. Reported specificities were 100% when detecting trisomy 21, trisomy 13, triploidy, and fetal sex, and 99.9% for trisomy 18 and monosomy X [6].
Why NIPT is clinically important
With the advent of SNP-based NIPT, the increase in the number of populations that can affordably and conveniently receive prenatal testing has dramatically increased and, subsequently, so has the identification of children with genetic abnormalities. Through early identification of chromosomal aneuploidies, children can receive early intervention services that are critical to the management of the associated disorders. This is especially true regarding the X and Y chromosomal variations that the NIPT identifies, specifically 47, XXY.
The impact of prenatal testing on 47, XXY
47, XXY (Klinefelter Syndrome) is characterized by the presence of an additional X chromosome and has a frequency of occurrence of 1 in 400 to 1 in 1,000 births [7]. However, due to their mild phenotypic presentation only 25% of boys with the disorder will ever be properly diagnosed. Boys with 47, XXY present neurocognitive deficits in language-based learning disabilities, atypical social development as well as reading disorders [8]. Musculoskeletal findings consist of decreased muscle tonus with joint laxity, pectus excavatum and pescavus. MRI brain imaging in individuals with 47, XXY revealed morphological, volumetric, and gray and white matter differences that are associated with the deficits in neurodevelopmental performance [9].
Androgen insufficiency in XXY has been described in several studies and it has been posited that the androgen deficiency contributes to the neurodevelopmental challenges associated with these disorders, as small research studies report improved brain function in association with androgen replacement [10]. Additionally, recent studies on 47, XXY and 49, XXXXY showed improvement in selected aspects of neurodevelopmental outcome when treated with androgen prior to 24 months of age [11, 12]. The area of greatest difficulty in the disorder is speech and language of which early hormonal treatment (EHT) has shown the most robust improvements in select areas of the verbal domain.
Boys with 47, XXY are susceptible to atypical social interactions, social isolation, and poor self-esteem as a result of the significant language-based learning disorders [9]. Ultimately, these issues may lead to low employment rates, depression and behavioural disruptions if not treated early in life [13]. Although there is a wide variability of cognitive capabilities in 47, XXY individuals, research studies indicate that prenatally diagnosed children demonstrate higher intellectual abilities [9]. Late diagnosis and untreated learning disorders coupled with deficits in executive function may result in significant neurocognitive challenges and behavioural disruptions [13]. School failure is common in these circumstances, which is costly for society in the form of low employment and high risk for psychiatric disturbances of depression and anxiety.
The importance of prenatal diagnosis is critical for the timely implementation of targeted and syndrome-specific treatments, most importantly EHT, and ensuring an optimal developmental trajectory for the child. The development of speech, language and early neurocognitive skills is critical to the growth of later reading proficiency and academic success. These skills are the building blocks for advanced abstract thinking capabilities and as a result allow for job employment and independent living. Research suggests that without timely treatment the growth of these critical neurodevelopmental abilities would be stunted or possibly altogether halted.
Summary
Although this article highlights only one disorder that can be identified through NIPT, the studies presented throughout demonstrate that the neurodevelopmental function of a very common neurogenetic disorder may be improved through early treatment. The importance of NIPT for early identification is imperative in XXY as well as other X and Y chromosomal disorders. The ramifications of prenatal detection and early identification cannot be understated; with knowledge comes the ability to improve a child’s life as well as the family’s well being from the moment of birth onward.
References
1. Invasive prenatal testing for aneuploidy. ACOG Practice Bulletin No. 88. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2007; 110: 1459–1467.
2. Noninvasive prenatal testing for fetal aneuploidy. Committee Opinion No. 545. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2012; 120: 1532–1534.
3. Lo YM, Tein JS, Lau TK, Haines CJ, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet. 1998; 62: 768–775.
4. Levy B, Norwitz E. Non-invasive prenatal aneuploidy testing: technologies and clinical implication. MLO Med Lab Obs 2013; 45: 8,10,12.
5. Samango-Sprouse C, Banjevic M, Ryan A, Sigurjonsson S, et al. SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy. Prenat Diagn. 2013; 33: 1–7.
6. Pergament E, McAdoo S, Curnow K, et al. SNP-based non-invasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y in a high- and low-risk cohort. Manuscript under review.
7. Morris JK, Alberman E, Scott C, Jacobs P. Is the prevalence of Klinefelter syndrome increasing? Eur J Hum Genet. 2008; 16: 163–170.
8. Samango-Sprouse CA, Gropman AL. Introduction: Past, present, and future care of individuals with XXY. Am J Med Genet C Semin Med Genet. 2013; 163C: 1–2.
9. Lee NR, Wallace GL, Clasen LS, Lenroot RK, et al. Executive function in young males with klinefelter (XXY) syndrome with and without comorbid attention-deficit/hyperactivity disorder. J Int Neuropsychol Soc. 2011; 22: 1–9.
10. Patwardhan AJ, Eliez S, Bender B, Linden MG, Reiss AL. Brain morphology in Klinefelter syndrome: extra X chromosome and testosterone supplementation. Neurology 2000; 54(12): 2218–2223.
11. Samango-Sprouse CA, Gropman AL, Sadeghin T, Kingery M, et al. Effects of short-course androgen therapy on the neurodevelopmental profile of infants and children with 49,XXXXY syndrome. Acta Paediatrica 2011; 100(6): 861–865.
12. Samango-Sprouse CA, Sadeghin T, Mitchell FL, Dixon T, et al. Positive effects of short course androgen therapy on the neurodevelopmental outcome in boys with 47, XXY syndrome at 36 and 72 months of age. Am J Med Genet A. 2013; 161A: 501–508.
13. Simpson JL, Graham JM, Samango-Sprouse CA, Swerdloff R. 2005. Klinefelter Syndrome. In Cassidy SB, Allanson JE (editors) Management of Genetic Syndromes, pp.323–334, 2nd edn. New York: Wiley-Liss.
The authors
Emily J. Stapleton1* BSc, Megan Hall2 PhD, and Carole A. Samango-Sprouse1, 3 EdD
1The Focus Foundation, Davidsonville, MD, USA.
2Natera Inc., San Carlos, CA, USA
3George Washington University of the Health Sciences, Washington, D.C., USA
*Corresponding author
E-mail: ndckids@gmail.com
Autoantibodies against MDA-5: very important serological markers in amyopathic dermatomyositis with rapidly progressive interstitial lung disease
by Assoc. Prof. Y. Muro, Assoc. Prof. K. Sugiura and Prof. M. Akiyama Autoantibodies against MDA-5 are serologically important biomarkers because they are mainly detected in patients with amyopathic dermatomyositis complicated with rapidly progressive interstitial lung disease (ILD). Anti-MDA-5 antibodies are useful not only for diagnosis but possibly also for monitoring disease activity in ILD.