Microsatellite instability, reflective of a defective mismatch repair system, has been implicated as one of the main pathways involved in the pathogenesis of colorectal carcinoma. Herein, we describe the role of the mismatch repair system in the development of colorectal carcinoma, the advantages and disadvantages of using immunohistochemistry as the primary method of determining mismatch repair status, and compare the suitability of colorectal endoscopic biopsy versus resection specimens as the testing material of choice.
by Dr Odharnaith O’Brien, Dr Éanna Ryan and Prof. Kieran Sheahan
Introduction
Owing to recent remarkable advances in our understanding of the molecular and genetic basis of disease, it is now known that colorectal carcinoma (CRC) is a heterogenous clinical entity characterized by multiple molecular subtypes [1]. One such molecular pathway involved in CRC pathogenesis is the microsatellite instability (MSI) pathway, where a deficient mismatch repair (dMMR) system leads to unchecked errors in DNA replication [2]. These errors result in a propensity for abnormal insertion or deletion of short, repetitive sequences of DNA (microsatellites), resulting in mutations in cancer-related genes and ultimately neoplasia. Up to 15–20% of colorectal carcinomas are of MSI phenotype. An inherited predisposition to dMMR cancers, particularly CRC, is present in Lynch syndrome, the most common heritable cancer syndrome. It is due to autosomal dominant mutations in four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) or more rarely by mutations in EPCAM, a gene upstream of MSH2. Patients present at an earlier age and have an increased incidence of synchronous and metachronous CRCs. Histologically, tumours are poorly differentiated, frequently exhibiting a mucinous or signet ring cell morphology. Tumour infiltrating lymphocytes are often prominent and a Crohn’s-like inflammatory response may be present at the tumour periphery. However, the majority of dMMR CRCs arise sporadically and are a result of MLH1 promoter hypermethylation. Unlike in Lynch syndrome, these tumours affect the right side of the colon, are diagnosed at advanced age and have a female preponderance. They are, however, histologically similar to Lynch syndrome CRCs. Mutation of the BRAF V600E gene is present in 60–70% of sporadic dMMR tumours and is almost never seen in Lynch syndrome. As such, incorporating BRAF and/or MLH1 methylation status into MMR diagnostic algorithms offers potential exclusion criteria for genetic testing [3–5].
Why is it important to identify dMMR in colorectal carcinoma?
Diagnosing a patient with a dMMR cancer has a number of advantages:
1. Identification of patients with Lynch syndrome
Once diagnosed, these patients benefit from increased surveillance, prophylactic aspirin therapy and more radical surgery in order to facilitate the prevention and/or early detection of potential tumours (both colonic and extracolonic) [5].
2. It provides prognostic information
Several studies have shown that dMMR CRC has a better prognosis than MMR proficient (pMMR) CRC. dMMR tumours are less likely to develop lymph node and liver metastases. However, in advanced disease (stage IV) dMMR status can portend a poorer prognosis [6–8].
3. It provides predictive information
dMMR tumours likely have a reduced response to 5-flurouracil based chemotherapy. In addition, advanced dMMR tumours have been shown to have a better response rate and progression free survival to the anti PD-1 drug pembrolizumab when compared to pMMR tumours [7–9].
Reliance by clinicians on clinical criteria such as the revised Bethesda guidelines to determine which patients should undergo screening for Lynch syndrome results in inaccurate determination of eligibility for screening in up to 28% of cases [10]. Consequently, a number of organizations have recently published guidelines endorsing reflex MMR testing of all diagnosed CRCs, including the National Institute for Health and Care Excellence (NICE), the American Society for Clinical Pathology (ASCP) and the American Society for Clinical Oncology (ASCP), among others [11–12]. The cost effectiveness of such a screening approach has been proven by several studies [13].
Diagnosis
Diagnosis of dMMR tumours is either via PCR amplification of specific microsatellite repeats in formalin-fixed, paraffin-embedded tumour tissue or by immunohistochemistry (IHC) which confirms the absence or presence of MMR proteins. Both MSI testing and IHC have virtually equivalent informative value in predicting germline mutation [3, 14]. Given that IHC is more widely available in general pathology laboratories and is a rapid, efficient and cost-effective method of testing, it is the more frequently used test. It also has the added benefit of directing germline testing to the particular mutated gene.
A number of commercially available MMR IHC antibodies are available for laboratory use. A protocol using a panel of four immunohistochemical antibodies to the four mismatch repair gene proteins (MLH1, MSH2, MSH6, PMS2) is recommended (Fig. 1). Complete loss of expression of one or more MMR protein is suggestive of dMMR. Loss of MLH1 often occurs in conjunction with loss of PMS2. This is due to the fact that MLH1 protein forms a heterodimer complex with PMS2. Isolated loss of PMS2 Is indicative of a defect in the PMS2 gene. However, combined loss of PMS2 and MLH1 indicates the defect lies in MLH1, as MLH1 confers stability to PMS2. A similar situation is seen with MSH2 and MSH6; isolated loss of MSH6 indicating defective MSH6, whereas loss of expression of both proteins indicates the defect involves MSH2. Background positive IHC staining in intratumoural lymphocytes or of adjacent normal colonic epithelium, if present, serve as reliable internal positive controls [5].
Once loss of expression of any IHC MMRP is confirmed, further testing is required. In cases where there is loss of MLH1, testing for the presence of BRAF V600E mutation and MLH1 hypermethylation, as mentioned previously, can further stratify those patients who likely have sporadic dMMR tumours. Patients demonstrating loss of MSH2, MSH6 or PMS2, and patients demonstrating loss of MLH1 who are BRAF V600E negative and MLH hypermethylation negative, should undergo germline testing to confirm Lynch syndrome (Fig. 2).
MMR IHC testing is typically performed on CRC resection specimens. Data has recently begun to accumulate that the yield of IHC testing performed on endoscopic biopsy material may be as good as that performed on surgical resections. We recently published a study evaluating the reliability of MMR IHC in CRC from preoperative endoscopic biopsy tissue when compared to matched surgical resection specimens and demonstrated 100% concordance in 53 cases of dMMR (n=10) and pMMR (n=43) tumours [14]. Our results corroborate the results of other studies that indicate endoscopic biopsies are a suitable source of tissue for MMR IHC analysis [15–17].
Preferential testing of MMR status on endoscopic biopsy samples over resection specimens carries a number of advantages. Immunostaining is highly sensitive to the degree of tissue fixation; given the small size of biopsy samples, faster and more thorough fixation may result in superior quality staining. Additionally, neoadjuvant chemoradiotherapy used in the standard treatment of locally advanced rectal tumours may result in a complete pathologic response, with no residual tumour available for testing. Neoadjuvant treatment can also occasionally alter the MMRP status of the tumour. In these two scenarios, the pretreatment biopsy could provide reliable testing material.
Endoscopic biopsies could also be used to initiate earlier and indeed preoperative genetic testing, allowing informed clinical decisions regarding the extent of resection to be made before surgery in those patients confirmed as having Lynch syndrome. The option of total colectomy as an alternative to segmental colectomy could be discussed, particularly with younger patients, to reduce the risk of metachronous CRC and the need for intense postoperative surveillance. In addition, females identified as having Lynch syndrome, who have completed their families, could be considered for concurrent hysterectomy, with/without bilateral salpingo-oophorectomy, in order to prevent the development of a gynecological tract malignancy and spare them a potential additional future procedure.
Recent studies suggest that dMMR tumours may respond well to immunotherapy in patients with advanced disease [9]. In the instance that an advanced tumour is inoperable at diagnosis, metastatic or endoscopic biopsy tissue could be used to screen for dMMR and Lynch syndrome, and direct immunotherapy.
Despite these advantages, some limitations exist in the use of IHC to determine MMR status which are not just specific to biopsy tissue. Rare missense mutations have been reported in MLH1 and MSH6 genes that affect MMR protein function but not translation and antigenicity – in this scenario the tumour harbours a defective protein, but one which demonstrates retention of IHC staining, giving a false result [19].
Intratumoural heterogeneity, where there is heterogeneity of MMR protein expression within a single tumour, also represents a potential pitfall [20]. This may be of particular concern in biopsy samples as they represent only a small proportion of a tumour and could erroneously misclassify the MMR status by virtue of inadequate sampling. Another issue is the small size of endoscopic biopsies; adequate material may not be available for IHC. Encouraging generous tumour sampling at the time of biopsy could reduce the risk of such limitations. Heterogeneity in the MMR status of CRC is rare and is thought in many instances to be a result of suboptimal tissue fixation. Given biopsies are usually of small size, adequate fixation of tissue can be assured.
Conclusion
Up to 15–20% of CRCs are of MSI phenotype, secondary to either sporadic methylation-induced silencing or inherited mutations in MMR-related genes. IHC is an effective and reliable testing modality for determining MMR status in CRC. Colorectal endoscopic biopsy and resection specimens are both suitable sources of testing material, with resection specimens currently the preferred specimen type. Endoscopic biopsy samples may become increasingly important as a testing material as the potential of tailored approaches to surgery, chemotherapy and immunotherapy becomes a standard of care in this era of personalized medicine.
References
1. Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, Marisa L, Roepman P, Nyamundanda G, et al. The consensus molecular subtypes of colorectal cancer. Nat Med 2015; 21(11): 1350–1356.
2. Poulogiannis G, Frayling IM, Arends MJ. 2010. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology 2010; 56(2): 167–179.
3. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, Walsh-Vockley C, Petersen GM, Walsh MD, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20(4): 1043–1048.
4. Bouzourene H, Hutter P, Losi L, Martin P, Benhattar J. Selection of patients with germline MLH1 methylation and BRAF mutation. Fam Cancer 2010; 9: 167–172.
5. Richman S. Deficient mismatch repair: read all about it (Review). Int J Oncol 2015; 47: 1189–1202.
6. Saridaki Z, Souglakos J, Georgoulias V. Prognostic and predictive significance of MSI in stages II/III colon cancer. World J. Gastroenterol 2014; 20(22): 6809–6814.
7. Guastadisegni C, Colafranceschi M, Ottini L, Dogliotti E. Microsatellite instability as a marker of prognosis and response to therapy: a meta-analysis of colorectal cancer survival data. Eur J Cancer 2010; 46(15): 2788–2798.
8. Mohan HM, Ryan E, Balasubramanian I, Kennelly R, Geraghty R, Sclafani F, Fennelly D, McDermott R, Ryan EJ, et al. Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer. Eur J Surg Oncol 2016; 42(11); 1680–1686.
9. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, Skora AD, Luber BS, Azad NS, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Eng J Med 2015; 372(26): 2509–2520.
10. Mukherjee A, McGarrity TJ, Ruggiero F, Koltun W, McKenna K, Poritz L, Baker MJ. The revised Bethesda guidelines: extent of utilization in a university hospital medical center with a cancer genetics program. Hered Cancer Clin Pract 2010; 8: 9.
11. Diagnostics guidance 27 (DG27). Molecular testing strategies for Lynch syndrome in people with colorectal cancer. NICE 2017 (https: //www.nice.org.uk/guidance/dg27).
12. Sepulveda AR, Hamilton SR, Allegra CJ, Grody W, Cushman-Vokoun AM, Funkhouser WK, Kopetz SE, Lieu C, Lindor NM, et al. ASCO, A. C. A. Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol 2017; 35: 1453–1486.
13. Snowsill T, Huxley N, Hoyle M, Jones-Hughes T, Coelho H, Cooper C, Frayling I, Hyde C. A systematic review and economic evaluation of diagnostic strategies for Lynch syndrome. Health Technol Assess 2014; 18(56): 1–406.
14. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Clendenning M, Sotamaa K, Prior T, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008; 26: 5783–5788.
15. O’Brien O, Ryan É, Creavin B, Kelly ME, Mohan HM, Geraghty R, Winter DC, Sheahan K. Correlation of immunohistochemical mismatch repair protein status between colorectal carcinoma endoscopic biopsy and resection specimens. J Clin Pathol 2018; 71(7): 631–636.
16. Kumarasinghe AP, de Boer B, Bateman AC, Kumarasinghe MP. DNA mismatch repair enzyme immunohistochemistry in colorectal cancer: a comparison of biopsy and resection material. Pathology 2010; 42(5): 414–420.
17. Warrier SK, Trainer AH, Lynch AC, Mitchell C, Hiscock R, Sawyer S, Boussioutas A, Heriot AG. Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy. Dis Colon Rectum 2011; 54(12): 1480–1487.
18. Vilkin A, Leibovici-Weissman Y, Halpern M, Morgenstern S, Brazovski E, Gingold-Belfer R, Wasserberg N, Brenner B, Niv Y, et al. Immunohistochemistry staining for mismatch repair proteins: the endoscopic biopsy material provides useful and coherent results. Hum Pathol 2015; 46(11): 1705–1711.
19. Klarskov L, Holck S, Bernstein I, Okkels H, Rambech E, Baldetorp B, Nilbert M. Challenges in the identification of MSH6-associated colorectal cancer: rectal location, less typical histology, and a subset with retained mismatch repair function. Am J Surg Pathol 2011; 35(9): 1391–1399.
20. Watson N, Grieu F, Morris M, Harvey J, Stewart C, Schofield L, Goldblatt J, Iacopetta B. Heterogeneous staining for mismatch repair proteins during population-based prescreening for hereditary nonpolyposis colorectal cancer. J Mol Diagn 2007; 9: 472–478.
The authors
Dr Odharnaith O’Brien* MB BCh BAO, Dr Éanna Ryan MB BCh BAO, and Prof. Kieran Sheahan MB BCh BAO
Department of Pathology, St. Vincent’s
University Hospital, Dublin, Ireland
*Corresponding author
E-mail: odharnaithobrien@ gmail.com
Gonotec: a continued success story since 1979
, /in Featured Articles /by 3wmediaDxH 900 hematology analyzer – the right results, the first time
, /in Featured Articles /by 3wmediaMonoclonal Antibodies
, /in Featured Articles /by 3wmediaLyme disease diagnosis: waiting for the next gold standard
, /in Featured Articles /by 3wmediaLyme disease is caused by Borrelia spirochaetes: predominantly Borrelia burgdorferi in North America (but also present in Europe), and predominantly B. afzelii and B. garinii in Europe and Asia and is spread to people via infected deer ticks. Infection occurs after only a minority of tick bites, but is typified by three stages. Stage 1, early localized lyme disease is characterized by the bull’s eye rash (erythema migrans (EM)). Stage 2, early disseminated infection occurs within days to weeks after the local infection as the bacteria begin to spread through the bloodstream. Stage 3, late disseminated infection, where the infection has spread throughout the body, can occur several months later in untreated or inadequately treated patients involving chronic symptoms that can be severe and disabling. Treatment by antibiotics is effective in the early localized stage of the disease but this is often hampered by late diagnosis. Diagnosis can be delayed for a number of reasons: there is a lack of awareness in the general public (as well as GPs outside of what are thought to be the high-risk areas); approximately 25% of people do not get the typical bull’s eye rash; and symptoms can be so varied and vague that, when occurring weeks or months later, are difficult to relate back to the time of the tick bite. Knowledge of a tick bite and an associated EM rash is sufficient for diagnosis. However, in cases where there is a clinical suspicion of Lyme disease but no EM rash, laboratory testing is advised. Testing for antibodies is done via a two-tiered approach, starting with a sensitive ELISA, which, if positive or equivocal, is followed by a more specific immunoblot. However, the overall sensitivity of the two-tiered tests is only 64% when done in the early stages of infection, which is when accurate diagnosis is most needed. Because of these diagnostic limitations, the prevalence of Lyme disease is likely to be far higher than is currently thought. With increasing incidence and geographic spread of the disease, better testing for diagnosis, particularly in the early stages of infection, is perhaps required. Research is ongoing into PCR methods as well as and for the detection of OspA antigens that are shed into urine. An LLT-MELISA (lymphocyte transformation test-memory lymphocyte immunostimulation assay) has been developed and is suggested to be a useful supportive diagnostic tool, particularly in infections acquired in Europe. In the USA, next-generation sequencing (NGS) has been used for specific pathogen identification and to guide treatment decisions. With technological advances making NGS quicker and cheaper, could this eventually become the next gold standard test for Lyme disease?
Urine ethyl glucuronide and ethyl sulphate measurement using liquid chromatography-tandem mass spectrometry
, /in Featured Articles /by 3wmediaBackground
Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are minor ethanol metabolites that can be used to detect recent alcohol consumption [1, 2]. Following the ingestion of alcohol, over 95% is metabolized by alcohol dehydrogenase to acetaldehyde. Up to 5% of ethanol is excreted unchanged in breath, sweat and urine. A small amount of ethanol (<0.1%) is metabolized in the liver by conjugation of glucuronic acid or sulphate to form EtG and EtS (Fig. 1). Following alcohol consumption, ethanol itself can only be detected in breath or urine for up to 6 or 12 hours, respectively (depending on the amount of alcohol consumed) [3]. In comparison, it has been demonstrated that EtG and EtS can be detected in urine for at least 24 hours and over 48 hours with heavy alcohol consumption [4].
The ability of these markers to detect alcohol intake over a longer time period means that they can be useful to identify alcohol relapses in alcohol-dependent individuals in treatment programmes [5]. In the UK, alcohol treatment programmes rely on breath ethanol and self-reporting to detect recent alcohol intake. However, this will only detect a proportion of individuals who are continuing to drink alcohol; this has been a low as 7% in one study comparing breathalyser/self-reported alcohol intake to urine EtG measurement [6]. Therefore, EtG and EtS can be helpful to detect those in alcohol treatment who are continuing to drink alcohol but deny it and have a negative breath ethanol test [7]. This allows additional interventions in individuals who are continuing to drink, which may ultimately improve outcomes. During 2016–17, 80 454 individuals entered alcohol treatment in England; of those 61% were free of alcohol dependence following the standard 12-week programme [8]. Therefore, improved detection of continuing alcohol consumption could lead to initiation of earlier intervention and altered strategies to increase the numbers successfully completing treatment.
Measurement of ethyl glucuronide and ethyl sulphate
Liquid chromatography (LC) to separate analytes with detection using mass spectrometry (MS) is now routinely used in clinical laboratories for an increasing number of tests. It is routine practice in urine toxicology testing for results to be confirmed by either LC or gas chromatography with detection using MS and it has been recommended by the United States Substance Abuse and Mental Health Services Administration (SAMHSA) that MS confirmation should be used for the measurement of EtG and EtS [9].
In tandem MS, two mass spectrometers are arranged sequentially with a ‘collision cell’ placed between the two instruments (Fig. 2). Using selective reaction monitoring, the first mass spectrometer (MS1) selects the ion with the mass/charge (m/z) ratio of interest. The selected ion (parent ion) is fragmented into small ions that enter the second mass spectrometer (MS2) where an ion with a specific m/z ratio is selected (daughter). Detection of analytes using an m/z ratio is very specific and sensitive allowing detection of very small amounts of EtG and EtS.
A number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for EtG and EtS have been published and a reference method has been proposed for EtG using solid phase extraction followed by LC-MS/MS [10]. Deuterated standards (EtG-d5 and EtS-d5) are readily available to purchase for use as internal standards ensuring reproducibility and reliability; an internal standard must mimic the analyte of interest but have a different mass to allow the MS detector to differentiate between the analyte of interest and the internal standard.
Sample preparation in published methods ranges from solid phase extraction to protein precipitation to dilution of urine in mobile phase. Solid phase extraction or protein precipitation of urine samples prior to LC-MS/MS can reduce the presence of potentially interfering substances which may cause ion suppression. It may also help to increase the lifespan of the column. For chromatographic separation of EtG and EtS, the mobile phases are usually formic acid in HPLC grade water and acetonitrile. Published methods have used both isocratic and gradients of mobile phase A and B to achieve separation of EtG and EtS; this is dependent on the sample preparation, the exact composition of the mobile phases and the column chosen. A rapid sample preparation of diluting urine samples in mobile phase A and then adding internal standard has been shown to be effective with no ion suppression or enhancement at or near the retention times for EtG and EtS [11]. Our experience has been to use an increasing gradient of mobile phase B (acetonitrile) from 1% to 10% over the first 2 minutes and then 10% to 100% from 2 minutes to 2.5 minutes. The increase from 1% to 10% acetonitrile elutes EtS/EtS-d5 at 1.27 minutes and the increase from 10% to 100% elutes EtG/EtG-d5 at 2.03 minutes. Figure 3 shows an example chromatogram for a urine sample collected from an individual attending the community based alcohol treatment programme; the high EtG and EtS results demonstrate that this person was continuing to drink alcohol.
Using MS to measure EtG and EtS requires the availability of LC-MS/MS equipment within the laboratory, the technical expertise required to set up an LC-MS/MS method and a dedicated member of staff to perform the analysis. In laboratories already using LC-MS/MS for other assays, there should be no difficulty in setting up a method to measure urine EtG and EtS.
An enzyme immunoassay method is also available to measure EtG and may be adapted for use on many automated laboratory analysers. This method has been shown to compare well to an LC-MS method [12]. For routine use, an immunoassay for EtG on an automated analyser has a number of advantages including rapid turnaround times, availability of EtG analysis out of routine working hours and the same staff members performing the analyses of multiple tests at the same time. However, there is no requirement for urine EtG and EtS analysis to be performed 24/7 as they would not be required in an acute setting. Generally, clients in a community treatment programme attend weekly, so once or twice weekly analysis using LC-MS/MS should be adequate for feedback of results to clients at their next visit. Not requiring a dedicated member of staff (as would be required for LC-MS/MS) is advantageous but according to SAMHSA guidelines, immunoassay results will require confirmation using a MS method. In addition, there is currently no immunoassay method available to measure EtS. This is important as there are a number of scenarios that can cause a false positive EtG result with a negative EtS result. For example, ‘positive’ EtG results (but not EtS results) have been demonstrated after the consumption of non-alcoholic beers (alcohol content 0.5%) [13]. EtG could also be formed in subjects with glycosuria and E.coli infection. If ethanol was formed due to the fermentation of sugars in the urine, this could be converted to EtG by bacteria present in the urine [14]. EtS would not be produced so again EtS can verify whether the EtG result is a true positive. Both EtG and EtS have been detected in individuals who used ethanol-based mouthwash or hand gel; however, the mouthwash was gargled 4 times/day which is much higher than the recommended frequency of use [15]. Owing to these factors, it is advisable to measure both EtG and EtS, which is currently only possible if using LC-MS/MS.
Cut-off values for EtG and EtS
There has been a lot of debate in the literature about suitable cut-off values to use for EtG and EtS. Some authors have suggested using the lower limit of detection (LLOD) or lower limit of quantitation (LLOQ) for the method so that any detectable EtG and EtS is a ‘positive’ result. However, the LLOD and LLOQ in LC-MS/MS methods will be variable between laboratories depending on a number of factors including sample preparation, column choice, chromatography and the tandem MS optimization. For EtG and EtS, the published LLOQs range from 0.05–0.20 mg/L and 0.04–0.10 mg/L respectively. New Clinical & Laboratory Standards Institute (CLSI) guidelines were published in 2016 and these should help to improve standardization between LC-MS/MS methods [16]. Alternatively, cut-off values could be defined by measuring EtG and EtS in a non-drinking population and incorporating measurement uncertainty (0.26 mg/L and 0.22 mg/L for EtG and EtS respectively) [11]. For EtG, a cut-off of 0.50 mg/L has been proposed to reduce the risk of false positive results. The disadvantage of a higher EtG cut-off is a reduction in sensitivity. Jatlow et al. demonstrated that using a 0.50 mg/L cut-off would only detect the intake of a low dose of alcohol 12 hours earlier (estimated blood alcohol 20 mg/dL) in 50% of participants. However, all participants had results above 0.10 mg/L and 0.20 mg/L after the same low alcohol dose 12 hours earlier [4]. SAMHSA have suggested separating EtG results into ‘high’ positive (>1.00 mg/L), ‘low’ positive (0.50–1.00 mg/L) and ‘very low’ positive (0.10–0.50 mg/L). They suggest that a ‘very low’ positive result may indicate previous heavy drinking (1–3 days ago), previous light drinking (12–36 hours ago) or ‘extraneous’ exposure [9].
Another consideration for urine EtG and EtS analysis is the dilution of urine samples; in urine toxicology testing, it is standard practice to measure creatinine to check the validity of a urine sample. There is limited data on the utility of EtG and EtS creatinine ratios. However, it is good practice to measure creatinine and question the validity of the EtG and EtS results if the creatinine is ≤2.0 mmol/L [17].
Conclusion
Urine EtG and EtS are valuable additional tools to detect recent alcohol intake in individuals undergoing treatment for alcohol dependence to ensure continued abstinence. Owing to the risk of false positive EtG results from unintentional exposure (e.g. non-alcoholic beer, urine infection with glycosuria, ethanol-based hand gel/mouthwash), the measurement of EtS in addition to EtG is recommended. An immunoassay is available for EtG but only MS allows the detection of both EtG and EtS to confidently confirm recent alcohol intake. There are a number of published methods for LC-MS/MS for EtG and EtS which are applicable for routine use in a clinical laboratory.
References
1. Dahl H, Stephanson N, Beck O, Helander A. Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide. J Anal Toxicol 2002; 26: 201–204.
2. Helander A, Beck O. Ethyl Sulphate – a metabolite of ethanol in humans and a potential biomarker of acute alcohol intake. J Anal Toxicol 2005; 29: 270–274.
3. Helander A, Beck O, Jones W. Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol and 5-hydroxytryptophol. Clin Chem 1996; 42: 618–624.
4. Jatlow P, Agro A, Wu R, Nadim H, Toll BA, Ralevski E, Nogueira C, Shi J, Dziura JD, et al. Ethylglucuronide and ethyl sulfate assays in clinical trials, interpretation and limitations: results of a dose ranging alcohol challenge study and two clinical trials. Alcohol Clin Exp Res. 2014; 38: 2056–2065.
5. Dahl H, Voltaire Carlsson A, Hillgren K, Helander A. Urinary ethyl glucuronide and ethyl sulphate for detection of recent drinking in an outpatient treatment program for alcohol and drug dependence. Alcohol Alcohol 2011; 46: 278–282.
6. Wetterling T, Dibbelt L, Wetterling G, Göder R, Wurst F, Margraf M, Junghanns K. Ethyl glucuronide (EtG): better than breathalyser or self-reports to detect covert short-term relapses into drinking. Alcohol Alcohol 2014; 49: 51–54.
7. Armer J, Gunawardana L, Allcock R. The performance of alcohol markers including ethyl glucuronide and ethyl sulphate to detect alcohol use in clients in a community alcohol treatment programme. Alcohol Alcohol 2017; 52: 29–34.
8. Knight J, Brand P, Willey P, van der Merwe J. Adult substance misuse statistics from the National Drug Treatment Monitoring System (NDTMS): 01 April 2016 – 31 March 2017. Public Health England 2017
(https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/658056/Adult-statistics-from-the-national-drug-treatment-monitoring-system-2016-2017.pdf).
9. The role of biomarkers in the treatment of alcohol use disorders. Substance Abuse and Mental Health Services Administration (SAMHSA) Advisory 2012; 11(2) (https://store.samhsa.gov/shin/content/SMA12-4686/SMA12-4686.pdf).
10. Helander A, Kenan N, Beck O. Comparison of analytical approaches for liquid chromatography/mass spectrometric determination of the alcohol biomarker ethyl glucuronide in urine. Rapid Commun Mass Spectrom 2010: 24: 1737–1743.
11. Armer J, Allcock R. Urine ethyl glucuronide and ethyl sulphate using liquid chromatography-tandem mass spectrometry in a routine clinical laboratory. Ann Clin Biochem 2017; 54: 60–68.
12. Bottcher M, Beck O, Helander A. Evaluation of a new immunoassay for urine ethyl glucuronide testing. Alcohol Alcohol 2008; 43: 46–48.
13. Thierauf A, Gnann H, Wohlfarth A, Auwärter V, Perdekamp MG, Buttler KJ, Wurst FM, Weinmann W. Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of “non-alcoholic” beer. Forensic Sci Int 2010; 202: 82–85.
14. Helander A, Ollson I, Dahl H. Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consumption. Clin Chem 2007; 53: 1855–1857.
15. Reisfield G, Goldberger B, Pesce A, Crews BO, Wilson GR, Teitelbaum SA, Bertholf RL. Ethyl glucuronide, ethyl sulfate, and ethanol in urine after intensive exposure to high ethanol content mouthwash. J Anal Toxicol 2011; 35: 264–268.
16. Lynch K. CLSI C62-A: a new standard for clinical mass spectrometry. Clin Chem 2016; 62(1): 24–29.
17. European guidelines for workplace drug testing in urine. European Workplace Drug Testing Society 2015 (http://www.ewdts.org/data/uploads/documents/ewdts-urine-guideline-2015-11-01-v2.0.pdf).
The authors
Jane Armer*1 BA MSc FRCPath and Rebecca Allcock2 BSc MSc FRCPath
1Department of Blood Sciences, East Lancashire Hospitals NHS Trust, Blackburn, UK
2Department of Clinical Biochemistry, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, UK
*Corresponding author
E-mail: jane.oakey@elht.nhs.uk
Use of an LC-MS/MS 13-steroid serum panel in the diagnosis of adrenocortical carcinoma
, /in Featured Articles /by 3wmediaLiquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly becoming the method of choice in the clinical laboratory for the measurement of low molecular weight analytes. The major advantage that LC-MS/MS possesses relative to conventional laboratory techniques such as immunoassay is its higher specificity (and often sensitivity, although this is compound specific) and its ability to measure multiple compounds in a single run (multiplexing). LC-MS/MS thus provides the opportunity for more accurate and precise biochemical diagnosis and monitoring of human disease. One example of the increasing adoption of LC-MS/MS by clinical laboratories is the measurement of steroid hormones in various matrices (serum, saliva, urine).
Steroid metabolism
All steroids share a cyclopentanoperhydrophenanthrene nucleus, with individual species varying according to the presence of different functional groups attached to this four-ring structure, as well as by the oxidation state of the rings. Cortisol structure is given as an example in Figure 1. In humans, the major sites of steroid hormone production are the adrenal gland and the gonads. Steroids are synthesized from cholesterol via a series of enzyme-catalysed steps (Fig. 2), which are under tight regulation in healthy individuals by feedback mechanisms involving the hypothalamus and anterior pituitary. Steroids have a wide range of physiological functions which are summarized in Table 1.
Adrenocortical carcinoma – a diagnostic challenge
There are many endocrine disorders that result in the improper synthesis of steroids, and one of the rarest and most severe is adrenocortical carcinoma (ACC). ACC is a malignancy of the adrenal cortex with an annual incidence of 1 or 2 cases per million [1]. The majority of ACC cases are sporadic and occur in the fifth or sixth decade of life and more commonly in women; although ACC can be associated with several familial syndromes including Li-Fraumeni, Beckwith-Wiedemann, Lynch syndrome and multiple endocrine neoplasia type 1 [2]. Functional steroid hormone-producing tumours occur in around two-thirds of cases [3], presenting with varied signs and symptoms of steroid overproduction, most commonly Cushing’s syndrome (cortisol excess) and hyperandrogenism. ACC can progress rapidly in some patients, therefore it is vital that it is distinguished from benign adrenal adenomas, as ACC has a 5-year survival rate of <50% [2]. A surgical cure is only possible if the carcinoma is detected in its localized stage, otherwise the median survival period is <15 months [4].
The diagnosis of ACC is challenging as there is no single diagnostic tool that is able to distinguish ACC from other adrenal masses, including benign adenomas with glucocorticoid or mineralocorticoid excess, phaeochromocytoma and non-functioning adenomas. Imaging alone is insufficient for diagnosis, as although patients with ACC almost always present with tumours ≥4 cm, the presence of a large mass only has a clinical specificity of 61% [5]. Additionally, whereas up to two-thirds of tumours are functional, less than half of ACC cases present with clinical signs of steroid overproduction [3], with a further proportion presenting with other symptoms including abdominal pain. However, a significant proportion are discovered incidentally [2].
The European Network for the Study of Adrenal Tumours (ENSAT) currently recommends that the initial biochemical work-up for suspected ACC includes measurement of serum cortisol (both basal and assessment of suppression after dexamethasone), dehydroepiandrostenedione sulphate (DHEAS), androstenedione, testosterone, 17-hydroxyprogesterone, estradiol and aldosterone (if the patient is hypokalemic or hypertensive). An alternative approach is to measure steroid metabolites in urine using gas chromatography-mass spectrometry (GC-MS); increases in the excretion of metabolites of the steroid precursors 11-deoxycortisol, 17-hydroxypregnenolone and pregnenolone have been shown to provide particularly high diagnostic utility in ACC. Unfortunately, urine steroid profiling is not commonly available in clinical laboratories owing to lengthy sample preparation and complex result interpretation. Further, serum 11-deoxycortisol, 17-hydroxypregnenolone or pregnenolone measurements are rarely performed either because of lack of demand, or specificity of the available immunoassays which may be subject to significant levels of cross-reactivity.
As a result of these limitations, the use of LC-MS/MS is increasingly being adopted to provide more specific steroid hormone measurements. An approach we have taken in our laboratory is to develop and fully evaluate a multiplexed LC-MS/MS method panelling 13 steroids in serum [6] to include many of the steroid synthetic pathway intermediates currently not available for ACC work-up.
Use of a serum steroid panel
The steroids included in our serum panel are highlighted in Figure 2 and are as follows:
Samples are prepared for analysis by an initial protein precipitation step to remove steroids from their binding proteins, followed by liquid-liquid extraction in order to cleanly extract the steroids from remaining matrix components. Prepared extracts are then analysed by LC-MS/MS in which steroids are first resolved on a reverse phase C18 column by gradient elution followed by MS/MS detection using positive atmospheric pressure chemical ionization (APCI) operated in multiple reaction monitoring mode. Chromatographic separation of several isobaric (same mass to charge ratio) steroids is essential, as is the use of deuterated internal standards for all steroids in the method.
When we applied our method to adrenal tumour samples [6], we were able to show that between 4 and 7 steroids were elevated in all ACC cases in comparison to non-ACC adrenal tumours where a maximum of 1–2 steroids were abnormal. The cortisol precursor 11-deoxycortisol was most useful in the discrimination between ACC and non-ACC adrenal lesions, whereas other steroids markedly elevated in ACC included 17-hydroxypregnenolone and pregnenolone. Indeed, all steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC. This validates the use of a panelling approach when investigating adrenal masses.
Our findings compare well with urine steroid profiling studies. Although urine steroid profiling using 24-hour collections may offer greater clinical sensitivity compared to a single blood measurement owing to diurnal rhythms of steroid production, urine measurements rely on accurately timed collections that are often performed incorrectly and are inconvenient to the patient. Advantages of our LC-MS/MS serum panel compared to urine steroid profiling by GC-MS include a less labour intensive sample preparation, as well as less expertise required for the interpretation of complex profiles, as the serum method only targets selected steroids rather than the large number of their metabolites in urine.
Use of our LC-MS/MS serum steroid panel in ACC patients has further demonstrated the limitations of assessing serum steroids by immunoassay. We observed evidence of notable interference in ACC patients in the cortisol, progesterone, 17-hydroxyprogesterone and androstenedione immunoassays, inferred to be due to elevated concentrations of structurally related steroid precursors.
Future work
Currently, our 13-steroid serum panel has been used to study a relatively small number of ACC patients (because of the rarity of the disease), and clearly larger prospective studies are required to more fully determine the diagnostic utility of our panel in ACC. Further work is also required to clarify the effects of age, sex and diurnal variation on serum steroid panelling; nonetheless the most useful markers of ACC are markedly elevated above variation attributable to these biological factors. In addition to the complexity of interpreting biomarker panels, it is not only important to consider specific reference ranges, but to also consider the patterns in results which require an omics-based analysis approach to interpretation. The challenge surrounding this, as well as the requirement for clear presentation and reporting of results to clinicians requires close involvement of clinical colleagues for the development and introduction of such testing strategies.
The analysis of steroid panels by LC-MS/MS can also undoubtedly be used in other conditions including inborn errors of steroid metabolism such as congenital adrenal hyperplasia (CAH) and polycystic ovarian syndrome (PCOS).
Although we have demonstrated the advantages of our LC-MS/MS steroid panel compared to routine immunoassays, there are undoubtedly disadvantages of using LC-MS/MS. These include the initial cost of instrument purchase, the increased expertise required and often a more laborious sample preparation. Additionally, the specificity of mass spectrometry should not be readily assumed; careful selection of multiple reaction monitoring (MRM) transitions and chromatography conditions are essential to separate isobaric steroids and other interfering compounds. However, in the context of improving the biochemical tools available to us to aid the diagnosis of ACC, the advantages of LC-MS/MS far outweigh these limitations.
Summary
In summary, LC-MS/MS serum steroid panelling offers an additional tool for the challenge that is the diagnosis of ACC. Our method combines measurement of both common and rarely measured steroids in a single sample, which we have shown provides useful data to aid the discrimination of ACC from benign adrenal tumours. Use of LC-MS/MS gives several advantages over the immunoassay and GC-MS-based methods currently used to assess steroid overproduction, but further work is required to demonstrate the full potential of its use in the diagnosis of ACC.
References
1. Fassnacht M, Kroiss M, Allolio B. Update in adrenocortical carcinoma. J Clin Endocrinol Metab 2013; 98: 4551–4564.
2. Else T, Kim AC, Sabolch A, Ramond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD. Adrenocortical carcinoma. Endocr Rev 2014; 35: 282–326.
3. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, Schneider P, Smith DJ, Stiekema H, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumours. J Clin Endocrinol Metab 2011; 96: 3775–3784.
4. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, et al. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012;366:2189–2197.
5. Hamrahian AH, Ioachimescu AG, Remer EM, Motta-Ramirez G, Bogabathina H, Levin HS, Reddy S, Gill IS, Siperstein A, Bravo EL. Clinical utility of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadenomas: Cleveland Clinical experience. J Clin Endocrinol Metab 2005; 90: 871–877.
6. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, Diaz-Cano S, Galata G, Schulte KM, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocortical carcinoma. Clin Chem 2017; 63: 1836–1846.
The authors
Victoria Treasure* MSc and Dr David Taylor PhD
Department of Clinical Biochemistry
(Viapath), King’s College Hospital NHS Foundation Trust, London, UK
*Corresponding author
E-mail: Victoria.treasure@nhs.net
Automating LC-MS/MS analysis for streamlined clinical testing workflows
, /in Featured Articles /by 3wmediaWithin clinical laboratories, however, LC-MS/MS methods are used across a relatively limited number of disciplines, most notably endocrinology, immunosuppressant and therapeutic drug monitoring, toxicology, new born screening, microbiology, as well as small molecule, peptide and protein marker analysis. This powerful technique brings many advantages to clinical workflows, enabling laboratory scientists to analyse multiple analytes with greater specificity and sensitivity than alternative methods, such as some immunoassays.
Despite its numerous benefits for patient care, LC-MS/MS technology has not been adopted across the wider clinical setting. One of the biggest barriers preventing its broader use has been the lack of commercially available automated systems that address the specific needs of the clinical laboratory.
The importance of fast turnaround times
Conventional LC-MS/MS workflows typically involve a large number of manual and time-consuming processes. Indeed, while advances in the performance of LC separation and MS analysis techniques mean that measurement acquisition steps now take a matter of minutes to complete, batching of samples, sample preparation, data analysis and equipment maintenance can significantly extend the length of time that must be invested in each sample run. Moreover, the burden associated with manual data entry can significantly lengthen timelines. To ensure quality, further data validation steps are required before final reporting, resulting in significant amount of time devoted to processes that do not add real value to operations.
Traditional LC-MS/MS methods also require users to ensure careful batching and multiple runs need scheduling at appropriate times, which may prove challenging when faced with shift patterns or a weekend testing service. Overall, the labor intensive LC-MS/MS workflows limit sample throughput, while requiring a high level of human input and incurring a significant operating expense. As such, these methods do not fit well with the working practices of the clinical laboratory.
The need for accurate analysis
Manual methods also leave measurements vulnerable to human error. Even when analyses are performed by the most experienced laboratory scientists, these multi-step workflows are susceptible to mistakes, omissions or even small variations in the way protocols are conducted. If errors are identified, repeat experiments are required to correct them. This can significantly add to the time taken to obtain clinically useful insights, prevent timely patient treatment decisions and even undermine confidence in the accuracy of findings.
Given the complexity of conventional LC-MS/MS workflows, and to reduce the potential of human error, the operation of these systems has traditionally been assigned to highly skilled scientists with specialist knowhow. High levels of expertise are also essential for sample preparation and data analysis. As a consequence, many clinical laboratories have been facing the need to train their personnel, which can place an additional burden on budgets and bandwidth.
Automated LC-MS/MS driving process optimization
Analytical methods within the clinical lab must be automated, reliable and provide walk-away capabilities to meet clinicians’ need for rapid turnaround of accurate results. By eliminating many of the error-prone and time-consuming manual steps involved in traditional workflows, fully automated, random access LC-MS/MS systems are well placed to simplify and accelerate the collection of high quality data. This ability to assess samples quickly, while maintaining a high level of accuracy, would be especially beneficial to those assays that involve more complex processes, since they could be streamlined and automated to simplify workflow.
Undoubtedly, the future of clinical analysis is trending towards the broader adoption of fully automated systems. Automation will greatly benefit LC-MS/MS workflows, making this powerful technique accessible for a wide range of clinical applications, without the need to create a new team of highly trained experts. Laboratories that are already performing clinical LC-MS/MS testing will also be able to better manage their highly trained experts and apply their talents to the development and early implementation of newer, more esoteric, high value analytes – expanding the laboratory’s overall service capabilities as a result. Furthermore, while the capital cost of currently available LC-MS/MS systems is relatively high, operational costs related to materials are actually low. If the volume of samples is high enough, then the economy of scale will make cost of ownership comparable to alternative clinical testing methods.
Labs already performing laboratory developed tests (LDTs) using LC-MS/MS may be more resistant to automation. The development and validation of MS assays takes a significant amount of time and expertise, so there may be concern over the impact that automation will have on their existing LDT protocols. However, automation will not be a limiting factor in a laboratory’s ability to develop and implement LDTs. Automation has the potential to reduce the need for highly trained staff to apply themselves to the repetitive tasks, allowing them to focus on the development of emerging, clinically needed LDTs.
Meeting the needs of clinical LC-MS/MS analysis
The need for an automated LC-MS/MS system that addresses the unique requirements of the clinical laboratory has led to the development of the new Thermo Scientific™ Cascadion™ SM Clinical Analyser*. Designed to eliminate, automate and simplify many of the manual processes involved in traditional LC-MS/MS workflows, the system gives users all of the power of this important technology in an easy to implement tool.
Owing to its random access capability, the Cascadion SM Clinical Analyser removes the need for long periods of batch loading, and instead facilitates continuous, uninterrupted operation for rapid turnaround of results. This is particularly important for the out-of-hours service and processing of STAT samples. Moreover, by minimizing the potential for human error, the technology is enabling the collection of accurate measurements, the first time around. When implemented in the clinical setting, this level of dependability is helping to accelerate clinical outcomes and deliver real value for clinical laboratories.
Furthermore, because the system can be operated by non-LC-MS/MS experts, experienced scientists have more time to work in other capacities. With more time back in their daily routine, this gives clinical researchers, for example, the opportunity to develop new tests to meet an urgent unmet need or to support better patient care. This level of ease-of-use and simplicity not only relates to run-to-run performance, it also extends to system maintenance too. A recent study from Argent Global Services has found that monthly maintenance takes approximately 18 minutes, meaning that significant amounts of time can be saved and put to better use.
Conclusion
LC-MS/MS systems offer clear benefits for clinical applications. However, the lack of automated systems has posed a barrier to their broader uptake in the clinical setting. Requiring expert operation and the investment of significant time and resources to ensure compatibility with sample preparation processes and data review and reporting systems, traditional technologies have, until now, not adequately addressed the needs of clinical laboratories.
Fully automated, random access LC-MS/MS technology, designed specifically for clinical use, is alleviating these pain points and enabling clinicians to benefit from quality results at high throughputs, while reducing the need to perform repetitive manual tasks. The impact of these systems is benefitting clinical laboratories, helping to improve operational efficiencies and ensure clinicians receive the results they need to make informed treatment decisions in a timely fashion.
*This product is IVD/CE-marked. Product is not 510(k) cleared and not yet available for sale in the U.S.
Reference
www.thermofisher.comZhang V & Rockwood A. “Impact of Automation on Mass Spectrometry”. Clinica Chimica Acta 450 (2015): 298-303.
Use of immunohistochemistry in the determination of mismatch repair status of colorectal carcinoma
, /in Featured Articles /by 3wmediaby Dr Odharnaith O’Brien, Dr Éanna Ryan and Prof. Kieran Sheahan
Introduction
Owing to recent remarkable advances in our understanding of the molecular and genetic basis of disease, it is now known that colorectal carcinoma (CRC) is a heterogenous clinical entity characterized by multiple molecular subtypes [1]. One such molecular pathway involved in CRC pathogenesis is the microsatellite instability (MSI) pathway, where a deficient mismatch repair (dMMR) system leads to unchecked errors in DNA replication [2]. These errors result in a propensity for abnormal insertion or deletion of short, repetitive sequences of DNA (microsatellites), resulting in mutations in cancer-related genes and ultimately neoplasia. Up to 15–20% of colorectal carcinomas are of MSI phenotype. An inherited predisposition to dMMR cancers, particularly CRC, is present in Lynch syndrome, the most common heritable cancer syndrome. It is due to autosomal dominant mutations in four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) or more rarely by mutations in EPCAM, a gene upstream of MSH2. Patients present at an earlier age and have an increased incidence of synchronous and metachronous CRCs. Histologically, tumours are poorly differentiated, frequently exhibiting a mucinous or signet ring cell morphology. Tumour infiltrating lymphocytes are often prominent and a Crohn’s-like inflammatory response may be present at the tumour periphery. However, the majority of dMMR CRCs arise sporadically and are a result of MLH1 promoter hypermethylation. Unlike in Lynch syndrome, these tumours affect the right side of the colon, are diagnosed at advanced age and have a female preponderance. They are, however, histologically similar to Lynch syndrome CRCs. Mutation of the BRAF V600E gene is present in 60–70% of sporadic dMMR tumours and is almost never seen in Lynch syndrome. As such, incorporating BRAF and/or MLH1 methylation status into MMR diagnostic algorithms offers potential exclusion criteria for genetic testing [3–5].
Why is it important to identify dMMR in colorectal carcinoma?
Diagnosing a patient with a dMMR cancer has a number of advantages:
1. Identification of patients with Lynch syndrome
Once diagnosed, these patients benefit from increased surveillance, prophylactic aspirin therapy and more radical surgery in order to facilitate the prevention and/or early detection of potential tumours (both colonic and extracolonic) [5].
2. It provides prognostic information
Several studies have shown that dMMR CRC has a better prognosis than MMR proficient (pMMR) CRC. dMMR tumours are less likely to develop lymph node and liver metastases. However, in advanced disease (stage IV) dMMR status can portend a poorer prognosis [6–8].
3. It provides predictive information
dMMR tumours likely have a reduced response to 5-flurouracil based chemotherapy. In addition, advanced dMMR tumours have been shown to have a better response rate and progression free survival to the anti PD-1 drug pembrolizumab when compared to pMMR tumours [7–9].
Reliance by clinicians on clinical criteria such as the revised Bethesda guidelines to determine which patients should undergo screening for Lynch syndrome results in inaccurate determination of eligibility for screening in up to 28% of cases [10]. Consequently, a number of organizations have recently published guidelines endorsing reflex MMR testing of all diagnosed CRCs, including the National Institute for Health and Care Excellence (NICE), the American Society for Clinical Pathology (ASCP) and the American Society for Clinical Oncology (ASCP), among others [11–12]. The cost effectiveness of such a screening approach has been proven by several studies [13].
Diagnosis
Diagnosis of dMMR tumours is either via PCR amplification of specific microsatellite repeats in formalin-fixed, paraffin-embedded tumour tissue or by immunohistochemistry (IHC) which confirms the absence or presence of MMR proteins. Both MSI testing and IHC have virtually equivalent informative value in predicting germline mutation [3, 14]. Given that IHC is more widely available in general pathology laboratories and is a rapid, efficient and cost-effective method of testing, it is the more frequently used test. It also has the added benefit of directing germline testing to the particular mutated gene.
A number of commercially available MMR IHC antibodies are available for laboratory use. A protocol using a panel of four immunohistochemical antibodies to the four mismatch repair gene proteins (MLH1, MSH2, MSH6, PMS2) is recommended (Fig. 1). Complete loss of expression of one or more MMR protein is suggestive of dMMR. Loss of MLH1 often occurs in conjunction with loss of PMS2. This is due to the fact that MLH1 protein forms a heterodimer complex with PMS2. Isolated loss of PMS2 Is indicative of a defect in the PMS2 gene. However, combined loss of PMS2 and MLH1 indicates the defect lies in MLH1, as MLH1 confers stability to PMS2. A similar situation is seen with MSH2 and MSH6; isolated loss of MSH6 indicating defective MSH6, whereas loss of expression of both proteins indicates the defect involves MSH2. Background positive IHC staining in intratumoural lymphocytes or of adjacent normal colonic epithelium, if present, serve as reliable internal positive controls [5].
Once loss of expression of any IHC MMRP is confirmed, further testing is required. In cases where there is loss of MLH1, testing for the presence of BRAF V600E mutation and MLH1 hypermethylation, as mentioned previously, can further stratify those patients who likely have sporadic dMMR tumours. Patients demonstrating loss of MSH2, MSH6 or PMS2, and patients demonstrating loss of MLH1 who are BRAF V600E negative and MLH hypermethylation negative, should undergo germline testing to confirm Lynch syndrome (Fig. 2).
MMR IHC testing is typically performed on CRC resection specimens. Data has recently begun to accumulate that the yield of IHC testing performed on endoscopic biopsy material may be as good as that performed on surgical resections. We recently published a study evaluating the reliability of MMR IHC in CRC from preoperative endoscopic biopsy tissue when compared to matched surgical resection specimens and demonstrated 100% concordance in 53 cases of dMMR (n=10) and pMMR (n=43) tumours [14]. Our results corroborate the results of other studies that indicate endoscopic biopsies are a suitable source of tissue for MMR IHC analysis [15–17].
Preferential testing of MMR status on endoscopic biopsy samples over resection specimens carries a number of advantages. Immunostaining is highly sensitive to the degree of tissue fixation; given the small size of biopsy samples, faster and more thorough fixation may result in superior quality staining. Additionally, neoadjuvant chemoradiotherapy used in the standard treatment of locally advanced rectal tumours may result in a complete pathologic response, with no residual tumour available for testing. Neoadjuvant treatment can also occasionally alter the MMRP status of the tumour. In these two scenarios, the pretreatment biopsy could provide reliable testing material.
Endoscopic biopsies could also be used to initiate earlier and indeed preoperative genetic testing, allowing informed clinical decisions regarding the extent of resection to be made before surgery in those patients confirmed as having Lynch syndrome. The option of total colectomy as an alternative to segmental colectomy could be discussed, particularly with younger patients, to reduce the risk of metachronous CRC and the need for intense postoperative surveillance. In addition, females identified as having Lynch syndrome, who have completed their families, could be considered for concurrent hysterectomy, with/without bilateral salpingo-oophorectomy, in order to prevent the development of a gynecological tract malignancy and spare them a potential additional future procedure.
Recent studies suggest that dMMR tumours may respond well to immunotherapy in patients with advanced disease [9]. In the instance that an advanced tumour is inoperable at diagnosis, metastatic or endoscopic biopsy tissue could be used to screen for dMMR and Lynch syndrome, and direct immunotherapy.
Despite these advantages, some limitations exist in the use of IHC to determine MMR status which are not just specific to biopsy tissue. Rare missense mutations have been reported in MLH1 and MSH6 genes that affect MMR protein function but not translation and antigenicity – in this scenario the tumour harbours a defective protein, but one which demonstrates retention of IHC staining, giving a false result [19].
Intratumoural heterogeneity, where there is heterogeneity of MMR protein expression within a single tumour, also represents a potential pitfall [20]. This may be of particular concern in biopsy samples as they represent only a small proportion of a tumour and could erroneously misclassify the MMR status by virtue of inadequate sampling. Another issue is the small size of endoscopic biopsies; adequate material may not be available for IHC. Encouraging generous tumour sampling at the time of biopsy could reduce the risk of such limitations. Heterogeneity in the MMR status of CRC is rare and is thought in many instances to be a result of suboptimal tissue fixation. Given biopsies are usually of small size, adequate fixation of tissue can be assured.
Conclusion
Up to 15–20% of CRCs are of MSI phenotype, secondary to either sporadic methylation-induced silencing or inherited mutations in MMR-related genes. IHC is an effective and reliable testing modality for determining MMR status in CRC. Colorectal endoscopic biopsy and resection specimens are both suitable sources of testing material, with resection specimens currently the preferred specimen type. Endoscopic biopsy samples may become increasingly important as a testing material as the potential of tailored approaches to surgery, chemotherapy and immunotherapy becomes a standard of care in this era of personalized medicine.
References
1. Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, Marisa L, Roepman P, Nyamundanda G, et al. The consensus molecular subtypes of colorectal cancer. Nat Med 2015; 21(11): 1350–1356.
2. Poulogiannis G, Frayling IM, Arends MJ. 2010. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology 2010; 56(2): 167–179.
3. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, Walsh-Vockley C, Petersen GM, Walsh MD, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20(4): 1043–1048.
4. Bouzourene H, Hutter P, Losi L, Martin P, Benhattar J. Selection of patients with germline MLH1 methylation and BRAF mutation. Fam Cancer 2010; 9: 167–172.
5. Richman S. Deficient mismatch repair: read all about it (Review). Int J Oncol 2015; 47: 1189–1202.
6. Saridaki Z, Souglakos J, Georgoulias V. Prognostic and predictive significance of MSI in stages II/III colon cancer. World J. Gastroenterol 2014; 20(22): 6809–6814.
7. Guastadisegni C, Colafranceschi M, Ottini L, Dogliotti E. Microsatellite instability as a marker of prognosis and response to therapy: a meta-analysis of colorectal cancer survival data. Eur J Cancer 2010; 46(15): 2788–2798.
8. Mohan HM, Ryan E, Balasubramanian I, Kennelly R, Geraghty R, Sclafani F, Fennelly D, McDermott R, Ryan EJ, et al. Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer. Eur J Surg Oncol 2016; 42(11); 1680–1686.
9. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, Skora AD, Luber BS, Azad NS, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Eng J Med 2015; 372(26): 2509–2520.
10. Mukherjee A, McGarrity TJ, Ruggiero F, Koltun W, McKenna K, Poritz L, Baker MJ. The revised Bethesda guidelines: extent of utilization in a university hospital medical center with a cancer genetics program. Hered Cancer Clin Pract 2010; 8: 9.
11. Diagnostics guidance 27 (DG27). Molecular testing strategies for Lynch syndrome in people with colorectal cancer. NICE 2017 (https: //www.nice.org.uk/guidance/dg27).
12. Sepulveda AR, Hamilton SR, Allegra CJ, Grody W, Cushman-Vokoun AM, Funkhouser WK, Kopetz SE, Lieu C, Lindor NM, et al. ASCO, A. C. A. Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol 2017; 35: 1453–1486.
13. Snowsill T, Huxley N, Hoyle M, Jones-Hughes T, Coelho H, Cooper C, Frayling I, Hyde C. A systematic review and economic evaluation of diagnostic strategies for Lynch syndrome. Health Technol Assess 2014; 18(56): 1–406.
14. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Clendenning M, Sotamaa K, Prior T, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008; 26: 5783–5788.
15. O’Brien O, Ryan É, Creavin B, Kelly ME, Mohan HM, Geraghty R, Winter DC, Sheahan K. Correlation of immunohistochemical mismatch repair protein status between colorectal carcinoma endoscopic biopsy and resection specimens. J Clin Pathol 2018; 71(7): 631–636.
16. Kumarasinghe AP, de Boer B, Bateman AC, Kumarasinghe MP. DNA mismatch repair enzyme immunohistochemistry in colorectal cancer: a comparison of biopsy and resection material. Pathology 2010; 42(5): 414–420.
17. Warrier SK, Trainer AH, Lynch AC, Mitchell C, Hiscock R, Sawyer S, Boussioutas A, Heriot AG. Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy. Dis Colon Rectum 2011; 54(12): 1480–1487.
18. Vilkin A, Leibovici-Weissman Y, Halpern M, Morgenstern S, Brazovski E, Gingold-Belfer R, Wasserberg N, Brenner B, Niv Y, et al. Immunohistochemistry staining for mismatch repair proteins: the endoscopic biopsy material provides useful and coherent results. Hum Pathol 2015; 46(11): 1705–1711.
19. Klarskov L, Holck S, Bernstein I, Okkels H, Rambech E, Baldetorp B, Nilbert M. Challenges in the identification of MSH6-associated colorectal cancer: rectal location, less typical histology, and a subset with retained mismatch repair function. Am J Surg Pathol 2011; 35(9): 1391–1399.
20. Watson N, Grieu F, Morris M, Harvey J, Stewart C, Schofield L, Goldblatt J, Iacopetta B. Heterogeneous staining for mismatch repair proteins during population-based prescreening for hereditary nonpolyposis colorectal cancer. J Mol Diagn 2007; 9: 472–478.
The authors
Dr Odharnaith O’Brien* MB BCh BAO, Dr Éanna Ryan MB BCh BAO, and Prof. Kieran Sheahan MB BCh BAO
Department of Pathology, St. Vincent’s
University Hospital, Dublin, Ireland
*Corresponding author
E-mail: odharnaithobrien@ gmail.com
Scientific literature review: Pathology
, /in Featured Articles /by 3wmediaCONCLUSION: The modified dithiothreitol method was able to reduce hemolysis during storage and to detect and identify alloantibodies in the presence of daratumumab.
METHODS: FS, imprecision, assay interference, limit of blank, linearity, and accuracy were assessed using the Abbott ARCHITECT i2000SR, SIEMENS ADVIA Centaur and IMMULITE 2000, Beckman Coulter DxI 800, and Roche MODULAR E170. Comparisons to an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were performed using patient samples from men, women, boys, and girls.
Competitive PCR-high resolution melting analysis: an improved approach to assess BRCA status in hereditary breast and ovarian cancer patients
, /in Featured Articles /by 3wmediaThe identification of BRCA pathogenic variants (PVs) is the major concern for the genetic counselling in families with a high risk of breast (BC) and ovarian cancer (OC). BRCA1 (breast cancer early onset 1) and BRCA2 (breast cancer early onset 2) are the two major susceptibility genes in BC/OC, conferring a lifetime risk up to 87% for BC and up to 44% for OC. BRCA mutations have been found in 4–14% of all OC, with a higher occurrence of about 22% in the high-grade serous OC [1]. The clinical relevance of the identification of BRCA PV carriers concerns many aspects of a patient’s evaluation. The first relevant implication is the assessment of the lifetime cancer risk. Additionally, BRCA testing has a relevant impact on the therapeutic approach and on the treatment outcomes owing to the possibility of selecting patients for biomarker-directed therapy based on the mutational status [2]. BRCA-positive patients with OC, particularly, respond well to platinum-based chemotherapy, especially in the high-grade serous OC subtype, and tend to retain platinum-sensitivity for longer than those without BRCA PVs. Additionally, the treatment with poly (ADP-ribose) polymerase (PARP) inhibitor (e.g. olaparib) was approved as a target therapy in patients with both germline and somatic BRCA PVs. PARP inhibitor therapy is able to improve progression-free survival in response to a recent platinum-based chemotherapy [3]. To date, licensed PARP inhibitor is part of the standard care and, consequently, BRCA evaluation is considered a routine investigation tool, useful before treatment management. With respect to these benefits, BRCA testing should be offered to all patients with OC on the basis of histological subtype, regardless of age, or family and personal history of malignancy. This issue causes an increase of the demand for BRCA testing with a strong challenge into the diagnostic laboratories committed in fulfilling the need of an efficient and rapid molecular evaluation [4].
The challenge of BRCA testing
To date 1700 PVs in BRCA1 and 1900 PVs in BRCA2 have been reported. Most of them are single nucleotide polymorphisms (SNPs) or small insertion-deletion mutations (indels), with a significant impact on the structure and function of the protein. Also large genomic rearrangements (LGRs), consisting mainly in large deletions or duplications, represent an important part of BRCA molecular lesions. To date, a total of 98 different BRCA LGRs have been reported, 81 in BRCA1 and 17 in BRCA2 [5] with a prevalence that varies considerably. Interestingly, deletion of BRCA1 exon 1a-2 is reported in several populations worldwide and is considered a recurrent BRCA LGRs in BC/OC patients [6]. Owing to the broad complexity in the mutational landscape of BRCA genes, comprehensive screening including the efficient assessment of both qualitative (SNPs, indels) and quantitative (LGRs) alterations is mandatory (Fig. 1). Diagnostic laboratories are adopting next-generation sequencing (NGS) technology for BRCA testing, which offers the potential of fast, cost-efficient and comprehensive sequencing. By choosing NGS technology, many considerations should be made, such as the selection of an NGS platform, including the enrichment methods, the sequencing chemistries, the analytical procedures and the variant calling for both germline and somatic PVs [2]. NGS is highly recommended as the reference sequencing method for BRCA testing because of the size of coding region and the method’s sensitivity in tumour sample evaluation. In fact, Sanger sequencing is not suitable for the analysis of somatic mutations, especially in samples where the percentage of tumour cells is under 50%, and it requires also a large amount of starting DNA [4]. Several methods are commonly used for LGR analysis, including multiplex ligation-dependent probe amplification (MLPA) and multiplex amplicon quantification (MAQ). However, these approaches are expensive and time-consuming, and consequently these are not always suitable for all laboratories. In this case, LGR evaluation of BRCA genes represents a bottleneck in terms of time and costs. In this context, the great benefit of the NGS approach is the opportunity to obtain both qualitative and quantitative information from the same sequencing data by using tailored bioinformatics algorithms [7]. Only a positive bioinformatics result needs to be confirmed using an alternative conventional method. In order to optimize our routine diagnostic procedures for BRCA testing, we recently developed a new molecular approach called competitive PCR-high resolution melting analysis (cPCR-HRMA) [8], as an alternative method for LGR identification in BRCA genes. HRMA is a simple and robust closed-tube method commonly used for diagnostics, forensic and research purposes. This method consists of a PCR amplification performed in the presence of saturating binding dyes followed by a melting reaction. Specifically, the incremental increase of the reaction temperature causes the denaturation of double-stranded DNA with the concomitant release of intercalated dye and a decrease of fluorescence signal. The specific sequence of the analysed amplicon, primarily relating to the GC content and the length, determines the melting behaviour observed in a fluorescence signal versus temperature plot. Additionally, the melting temperature (Tm) may be calculated as the derivative of the melting curve. The shape of the curves and the specific Tm value obtained in the output plots is used for the genotyping. The advantages of this technique include rapid turn-around times and a closed-system environment that decrease the risk of laboratory contamination [9].
cPCR-HRMA for LGR evaluation
HRMA technology is typically applied to detect a single substitution, as well as small indels variants [6, 10]. The new cPCR-HRMA represents an optimized HRMA method that allows an efficient evaluation of BRCA1 copy number variation (CNV) by relying on the melting behaviour of target BRCA amplicon compared to a reference amplicon in the same HRMA reaction. In particular, specific albumin sequences were chosen as unchanged CNV references and analysed by coupling them with specific BRCA1 exons in a duplex PCR assay preceding the melting analyses. The landmarks of this new HRMA rely on the primers and the amplification protocol design. First of all, primer pairs for the simultaneous amplification of target and reference sequences are selected in order to produce paired amplicons with comparable lengths (similar amplification efficiencies) and different melting temperatures (no overlap between amplicons melting peaks). Furthermore, the primer concentration used for both target and reference amplification was set in order to produce comparable PCR performance between the two amplicon types and to obtain melting profiles more suggestive of CNV. In addition, the PCR thermal cycling was carried on until the exponential phase, in which the amplification performance reflects the CNV status of the target region. These optimized features lead to melting profiles specifically tailored for CNV investigations allowing a rapid detection of samples affected by a change in copy number. Genotype association was assessed by direct interpretation of melting profiles, as shown in Figure 2: samples with similar profiles were clustered into the same genotype group and CNV positive samples showed a typical melting profile with a detectable shape comparing to the wild type. In addition to the qualitative evaluation, we provide also a semi-quantitative analysis of melting behaviour with the calculation of the fluorescence peak height ratio (R) of target the amplicon (BRCA1) to the reference amplicon (albumin), according to the formula:
The mean and the standard deviation of the R values calculated in a consistent number of control CNV samples allowed the identification of the reference range for the R parameter: WT sample (mean±2SD; 2 copies), deletion (≤mean−2SD; n copies) and duplication (≥mean+2SD; 3n copies). The R value calculated in each analysed sample is normalized with the average of the ratios calculated in the WT sample group, obtaining the normalized fluorescence peak height ratio (Rn). The latter is compared to the reference range in order to obtain the copy number interpretation. Taken together, the qualitative and the semi-quantitative evaluations of the cPCR-HRMA assay allow the correct identification of copy number status in BRCA gene, resulting as a rapid and alternative method for the analysis of LGRs. Advantages of cPCR-HRMA are the ease and fast handling of samples. Furthermore, this application needs the same reagents and equipment for standard HRMA protocols commonly used in many laboratories routines. By introducing this efficient alternative method, our first aim was the optimization of the BRCA workflow, promoting a more rational use of confirmatory testing, such as MLPA and MAQ. Finally, we are confident that a general implementation of BRCA testing is now necessary as an emerging challenge. After a complete genetic counselling and a multidisciplinary activity that involves geneticists, oncologist and all other professionals, the patient should be directed to specialized laboratories. The complexity of the potential BRCA mutations, coupled with their clinical relevance, leads to the mandatory adoption of a comprehensive molecular workflow for BRCA analysis that must be characterized by a low wait-time and efficient clinical reporting in order to guarantee a useful medical application.
References
1. Antoniou A, Pharoah PD, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003; 72(5): 1117–1130.
2. Capoluongo E, Ellison G, López-Guerreroc JA, et al. Guidance statement on BRCA1/2 tumor testing in ovarian cancer patients. Semin Oncol 2017; 44(3): 187–197.
3. George A, Kaye S, Banerjee S. Delivering widespread BRCA testing and PARP inhibition to patients with ovarian cancer. Nat Rev Clin Oncol 2017; 14(5): 284–296.
4. Capoluongo E, Scambia G, Nabholtz JM. Main implications related to the switch to BRCA1/2 tumor testing in ovarian cancer patients: a proposal of a consensus. Oncotarget 2018; 9(28): 19463–19468.
5. Sluiter MD, van Rensburg EJ. Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation. Breast Cancer Res Treat 2011; 125: 325–349.
6. Mazoyer S. Genomic rearrangements in the BRCA1 and BRCA2 genes. Hum Mutat 2005; 25(5): 415–422.
7. Scaglione GL, Concolino P, De Bonis M, et al. A whole germline BRCA2 gene deletion: how to learn from CNV in silico analysis. Int J Mol Sci 2018; 19(4): pii: E961.
8. Minucci A, De Paolis E, Concolino P, et al. Competitive PCR-high resolution melting analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: a new approach to assess quantitative status of BRCA1 gene in a reference laboratory. Clin Chim Acta 2017; 470: 83–92.
9. Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 2008; 85(1): 50–58.
10. De Paolis E, Minucci A, De Bonis M, et al. A rapid screening of a recurrent CYP24A1 pathogenic variant opens the way to molecular testing for idiopathic infantile hypercalcemia (IIH). Clin Chim Acta. (2018) Mar 21; 482: 8–13.
The authors
Elisa De Paolis MSc, Angelo Minucci PhD, Giovanni Luca Scaglione PhD, Maria De Bonis MSc, Ettore Capoluongo* PhD
Catholic University of The Sacred Heart, Rome, Italy
*Corresponding author
E-mail: ettoredomenico.capoluongo@policlinicogemelli.it