Screening for cancer: are biomarkers of value?

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– February/March 2011 28 Molecular diagnostics germline sequencing and insertion/deletion analysis of the MMR genes. If the tumour has been previously analysed by immunohistochemistry, targeted germline sequencing and insertion/deletion analysis of the gene corresponding to the missing MMR protein can be performed. Pre-analytic and analytic considerations of molecular diagnostic methods Genetic testing of solid tissue specimens presents a unique challenge for the clinical laboratory. Samples vary widely in tissue quantity (e.g. fine needle biopsy vs. resection specimen), quality (e.g. fresh frozen vs. formalin-fixed for 72 hours), and heterogeneity of tumour and non-tumour tissue. Due to these factors, highly robust and sensitive methods that can detect a clinically significant variant present in a low proportion of the sample are needed. Traditional Sanger sequencing has a sensitivity of only ~15-20% to detect minor allele populations within a mixture, which can result in false negative results for KRAS testing [29]. Pyrosequencing and melting curve analysis generally have better sensitivity, at ~1-10% [29]. Newer methods that increase sensitivity further to ~0.1-1% include co-amplification at lower denaturation temperature (COLD)- PCR, and peptide nucleic acid (PNA) clamping [30, 31]. Such sensitive methods have been described in the literature for KRAS [30-32] and BRAF [32] mutation detection in CRC, and have been shown to improve diagnostic accuracy. Additional techniques which could allow ultrasensitive detection of somatic mutations include next-generation sequencing [33] and limiting-dilution-PCR [34]. Importantly, with the increased ability to detect minor mutant populations within the sample, it will be necessary to determine the clinically relevant threshold in addition to the absolute analytical threshold. Conclusions and recommendations Marked improvements in our understanding of CRC biology over the last few years have led to the discovery of several genetic biomarkers that guide treatment and establish prognosis of patients with CRC. Although many biomarkers have been studied, currently only KRAS, BRAF, and MSI are sufficiently validated to support routine clinical use. Cases in which MSI is suspected based on clinicopathologic data should be tested. If MSI is demonstrated, the distinction between Lynch syndrome and sporadic CRC is fundamental to determine the necessity of genetic counselling and close oncologic surveillance. Sporadic CRC cases should be evaluated for KRAS and possibly BRAF if anti-EGFR therapy is an option. In the future, testing the PI3K pathway genes PIK3CA and PTEN may help define the population which is most likely to benefit from the expensive anti- EGFR treatment. The performance of the molecular methods used for the above determinations needs to be considered and clearly communicated so that patients receive the most accurate and meaningful information, and do not over-interpret the laboratory data. As the era of molecular oncology and personalised medicine unfolds, many changes in the way cancer is currently diagnosed and treated are expected to occur. Laboratory directors and staff alike have the responsibility of keeping up with the pace of change to continue offering the best diagnostic testing to clinicians and patients. References 1. Karsa LV et al. Best Pract Res Clin Gastroenterol 2010; 24(4): 381-396. 2. Boland CR et al. Gastroenterology 2010; 138(6):2073-2087.e2073. 3. Vilar E et al. Nat Rev Clin Oncol 2010; 7(3):153-162. Biomarker Freq. Detection Method Current Uses Possible Future Uses KRAS codon 12/13 mutations 40% DNA mutation testing Anti-EGFR resistance Prognosis BRAF V600E mutation 10% DNA mutation testing Microsatellite instability (MSI) 15% PCR/sizing Anti-EGFR resistance,Lynch/ HNPCC diagnosis Lynch/HNPCC diagnosis PIK3CA mutations 20% DNA mutation testing None BRAF inhibitor sensitivity Prognosis,5-FU resistance,Irinotecan sensitivity Anti-EGFR resistance,PI3K inhibitor sensitivity PTEN loss 30% IHC None Anti-EGFR resistance 18q loss 50% FISH, cytogenetics None Prognosis Topoisomerase 1 low 50% IHC None Irinotecan sensitivity Freq.: Frequency in colorectal cancer; 5-FU: 5-fluorouracil; IHC: Immunohistochemistry; FISH: Fluorescent in situ hybridization Table 1. Genetic Biomarkers in colorectal cancer. 4. Aaltonen LA et al. N Engl J Med 1998; 338(21): 1481-1487. 5. Hampel H et al. N Engl J Med 2005; 352(18):1851-1860. 6. Hampel H et al. J Clin Oncol 2008; 26(35):5783-5788. 7. Ward R et al. Gut 2001; 48(6):821-829. 8. Popat S et al. J Clin Oncol 2005; 23(3):609-618. 9. Kane MF et al. Cancer Res 1997; 57(5):808-811. 10. Fallik D et al. Cancer Res 2003; 63(18):5738-5744. 11. Bertagnolli MM et al. J Clin Oncol 2009; 27(11): 1814-1821. 12. Rajagopalan H et al. Nature 2002; 418(6901):934. 13. Bokemeyer C et al. J Clin Oncol 2009; 27(5):663-671. 14. Benvenuti S et al. Cancer Res 2007; 67(6):2643-2648. 15. Van Cutsem E et al. J Clin Oncol 2007; 25(13): 1658-1664. 16. Amado RG et al. J Clin Oncol 2008; 26(10):1626-1634. 17. Normanno N et al. Nat Rev Clin Oncol 2009; 6(9): 519-527. 18. Roth AD et al. J Clin Oncol 2010; 28(3):466-474. 19. Di Nicolantonio F et al. J Clin Oncol 2008; 26(35): 5705-5712. 20. Jhawer M et al. Cancer Res 2008; 68(6):1953-1961. 21. Sartore-Bianchi A et al. Cancer Res 2009; 69(5): 1851-1857. 22. Janku F et al. Mol Cancer Ther 2011. 23. Popat S et al. Eur J Cancer 2005; 41(14):2060-2070. 24. Braun MS et al. J Clin Oncol 2008; 26(16):2690-2698. 25. Greenson JK et al. Am J Surg Pathol 2003; 27(5): 563-570. 26. Laghi L et al. Oncogene 2008; 27(49):6313-6321. 27. Boland CR et al. Cancer Res 1998; 58(22):5248-5257. 28. Shia J. J Mol Diagn 2008; 10(4):293-300. 29. Tsiatis AC et al. J Mol Diagn 2010; 12(4):425-432. 30. Milbury CA et al. Clin Chem 2009; 55(12):2130-2143. 31. Pritchard CC et al. BMC Clin Pathol England 2010; 10: 6. 32. Mancini I et al. J Mol Diagn 2010; 12(5):705-711. 33. Mardis ER et al. Hum Mol Genet 2009; 18(R2): R163-168. 34. Pohl G et al. Expert Rev Mol Diagn 2004; 4(1):41-47 The authors Carlos J Suarez, Eric Q. Konnick and Colin Pritchard Department of Laboratory Medicine University of Washington Seattle, WA 98195, USA The authors contributed equally to this work. Corresponding author: Colin C. Pritchard MD, PhD University of Washington Department of Laboratory Medicine Molecular Diagnostics Laboratory, Room NW-275 Seattle, WA 98195-7110, USA Tel. +1 206 598 6131 Fax +1 206 598 6189 e-mail: cpritch@uw.edu.
Microbiology 29 – February/March 2011 Laboratory diagnosis of syphilis: indirect detection methods Due to diverse clinical manifestations which are shared with other treponemal and non-treponemal diseases, it is important to diagnose syphilis with the aid of appropriate and reliable laboratory tests. by Dr Meghna Patel Syphilis is a sexually transmitted disease caused by the spirochete, Treponema pallidum [1, 2]. It is a chronic disease with many very serious complications, especially during pregnancy, affecting the cardio-vascular and nervous systems in particular. It can also facilitate the transmission of HIV [2, 3]. During the course of the disease there are diverse clinical manifestations, which are indistinguishable from those of many other diseases [1, 4]. In recent years the incidence of syphilis has greatly increased in the United States and in Germany [1, 5]. Syphilis is easy to cure in the early stage, but can become disabling in the later stages and can ultimately be fatal [1]. The disease progresses through four stages: the primary, secondary, latent and tertiary stages. The stage of the disease at which the patient presents has implications for diagnosis and treatment [6]. There are several laboratory tests, which can be categorised into direct and indirect detection of T. pallidum, to diagnose syphilis at the various stages of the disease. This article will focus on indirect detection methods; with direct methods there are difficulties in cultivation and maintenance of T. pallidum in the laboratory, and in samples from patients with the latent and late stages of the disease, there is a paucity of spirochetes for direct observation. However a good number of indirect detection methods have been developed and are routinely used to diagnose syphilis. Broadly these are classified as non treponemal and treponemal tests. Non treponemal tests These tests are often referred to as non specific, reagin or cardiolipin tests, as they use standardised antigen containing cardiolipin, cholesterol and lecithin to detect the presence of IgG and IgM antibodies to lipoidal material released from host cells [4]. US CDCapproved standard tests include the Venereal Diseases Research Laboratory (VDRL) slide test, the Rapid Plasma Reagin (RPR) card test, the Unheated Serum Reagin (USR) test and the Toluidine Red Unheated Serum Test (TRUST) [6]. Serum is preferred over plasma for both treponemal and non treponemal tests, however plasma can also be used for the RPR test and the TRUST. The VDRL and USR tests are microflocculation tests that are read using a microscope. Even though the VDRL is the only test directly applicable for CSF testing, its utility is limited because the antigenic suspension is unstable and must be freshly prepared every day for reliable results, and the serum sample must be heat-inactivated. However, the USR uses a very stable antigenic suspension and an unheated serum sample, which facilitates use. Being macroscopic flocculation tests, a microscope is not required to perform the RPR and TRUST. The RPR uses a stabilised VDRL antigen containing charcoal particles to facilitate reading of the test. In the TRUST, charcoal is replaced by toluidine red dye that gives pink coloured floccules with positive samples. These tests can be used as both qualitative and semi-quantitative assays to determine the titre of the sample. Non treponemal tests are of great value for monitoring the course of the disease during and after treatment, and for diagnosis of reinfection and relapse. In spite of these advantages, non treponemal tests cannot be used alone for syphilis diagnosis as they have limited sensitivity, especially in primary and late latent syphilis patients, and the prozone phenomenon in secondary syphilis can result in false negatives. False positives can also occur due to cross reactivity in patients with hepatitis, chicken pox, infectious mononucleosis, measles, malaria and connective tissue disease, as well as in pregnancy [4]. Span Diagnostic’s non treponemal tests RPR and TRUST provide stable, aqueous ready to use reagents with standardised immunoreactivity. They offer simple, rapid and inexpensive test for syphilis and are even suitable for epidemiological and field studies. The company has further improved its non treponemal tests with the use of synthetic VDRL antigen, manufactured by US-FDA approved technology, licensed from the CDC. Synthetic VDRL antigen offers better stability compared to natural VDRL antigen. Treponemal tests These are sometimes referred to as specific tests because they use T. pallidum or its components as antigen/s. Treponemal tests are mainly used for confirming the results of non treponemal tests that have low specificity. They are also important for detecting very early, congenital or late cases of syphilis when non treponemal tests are negative. However the usefulness of treponemal tests is limited by their cost and complexity, as well as by persistent positive results even in patients who have been successfully treated. In treponemal tests, anti-treponemal antibodies are detected by techniques such as agglutination, Enzyme Immuno Assay (EIA), immunofluorescence, immunoblotting and immunochromatogrphy. Indirect Agglutination tests The Treponema Pallidum HaemAgglutination (TPHA) and Treponema Pallidum Particle Agglutination (TPPA) tests are examples of indirect agglutination tests. In the TPHA classically sheep RBCs are coated with sonicated T. pallidum preparations. In positive samples these are agglutinated by the anti treponemal antibodies present. The TPHA is a highly sensitive test in all stages of the disease except in early primary syphilis. It is also reasonably specific but there are some false positives, from the use of sheep RBCs, in patients suffering from infectious mononucleosis, leprosy, collagen disease and other miscellaneous conditions; false positives are rare in healthy individuals [4]. The use of chicken, turkey and more recently human ‘O’ RBCs has successfully reduced the number of false positive results, as has the use of coloured gelatine or polymer particles, which has also greatly decreased the complexity of the test [2]. The TPPA test is an appropriate substitute for the microhaemagglutination assay (MHA-TP), as it is as sensitive as the fluorescent treponemal antibody absorbed (FTA- ABS) test for primary syphilis and as useful as the RPR for screening blood donors [7]. Furthermore it has been reported that sensitivity has increased due to the improved IgM binding capacity of sensitised gel particles. Enzyme Immuno Assay (EIA) The indirect competitive capture EIA, detecting IgG and/or IgM treponemal antibodies, has been developed by numerous laboratories. The tests have similar or higher sensitivity and
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