Effective screening strategies have not yet been developed for the early detection of ovarian cancer. The serum biomarker CA125, routinely used to aid diagnosis and monitor treatment response, is not informative in all patients. Recent analytical developments have prioritized promising candidate novel biomarkers or multi-biomarker panels for future clinical evaluation.
by E. L. Joseph, Dr M. J. Ferguson and Dr G. Smith
Introduction to ovarian cancer
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and the fifth leading cause of cancer related death among women with around 140 000 annual deaths worldwide. EOC can develop as one of four histotypes with the serous histotype being the most common and most aggressive. The remaining three non-serous histotypes, endometrioid, clear cell and mucinous cancers present less frequently. High grade serous ovarian cancer, heterogeneous in nature and rapidly progressive, has a poor prognosis, where a major contributing factor is the lack of ability to diagnose the disease at a sufficiently early stage to facilitate curative surgery. The 5-year survival rate is less than 30% for patients presenting with advanced disease spread beyond the ovaries (FIGO Stage 3/4), but if detected earlier combination therapy of cytoreductive surgery and adjuvant or neo-adjuvant chemotherapy with platinum and taxane-based drugs has the potential to cure 90% of patients [1]. Consequently the identification of biomarkers capable of detecting ovarian cancer at the earliest stages and monitoring disease progression are inherently important in tackling this lethal disease. Due to its prevalence, the ideal biomarker for detecting early stage ovarian cancer requires an extremely high specificity (>99%) and a minimum sensitivity of 75% [2]. Despite extensive research, no optimal ovarian cancer biomarker has yet been identified and such high specificity is unlikely to be met by a single agent. Many promising candidate biomarkers are however currently undergoing evaluation in clinical trials.
CA125
The only ovarian cancer biomarker routinely used in the clinic is cancer antigen 125 (CA125; mucin 16) currently considered the ‘gold standard’ cancer biomarker despite its limitations. In the majority of patients with EOC, expression of the CA125 glycoprotein is raised above the normal reference range (>35 U/ml blood), but it only has a sensitivity of 50% to 60% with a specificity of 90% in early stage postmenopausal patients [3]. Several factors, however, limit the utility of CA125 in routine population screening: it is not expressed in 20% of ovarian cancers, is only significantly elevated in 47% of early stage ovarian cancers (although increasing to 80–90% in advanced stage cancers) and can be raised in many benign conditions including endometriosis and peritonitis. Variability in CA125 expression throughout the menstrual cycle and in pregnancy is also a common confounding issue. A major clinical utility of CA125, however, is related to its ability to commonly reflect clinical response following chemotherapy treatment and as such is often successfully used to monitor a patient’s progress through chemotherapy. A reduction in CA125 expression during treatment is considered a positive prognostic outcome for the patient and serial serum measurements are currently used to predict therapeutic outcomes and estimate stability of the disease (Fig. 1).
Biomarkers under evaluation
There have now been multiple attempts to identify novel ovarian cancer biomarkers with varying success. The most promising serum biomarkers include HE4 and mesothelin.
HE4
Human epididymis protein 4 (HE4) has been shown to be consistently elevated above the normal level (151 pM) in ovarian cancers, with sensitivity of 95% and specificity of 73%. HE4 is differentially expressed in specific subtypes of ovarian cancer, potentially allowing clinicians to distinguish histotypes to aid treatment; HE4 was found to be overexpressed in 100% of endometrioid cancers, 93% of serous cancers but only 50% of clear cell cancers [4]. Unlike CA125, it is less likely to produce false positives in benign masses and it has also been proposed to be the best candidate biomarker for early detection of Stage I disease despite sensitivity and specificity of 46% and 95% respectively [2]. HE4 has recently obtained FDA approval in the USA for monitoring recurrence or progression of EOC and, in comparative tests, has been found to be superior to CA125 in classifying benign and borderline ovarian cancers.
Mesothelin
Mesothelin is a glycoprotein expressed by mesothelial cells, the expression of which has been found to be raised in mesothelioma, pancreatic and ovarian cancers.
It can be easily measured in both urine and serum, highlighting its potential as a non-invasive biomarker. Serum mesothelin levels were found to be increased in approximately 60% of ovarian cancers with 98% specificity.
One study found elevation of mesothelin in 42% of urine assays as opposed to 12% serum assays of early stage EOCs at 95% specificity which reinforces the potential of this glycoprotein as an early detection biomarker and the use of urine in preference to serum [2]. Higher levels of mesothelin were also found to be associated with poorer overall survival in patients following optimal debulking surgery or who have advanced stage ovarian cancer. A recent study, however, revealed that lifestyle choices such as smoking and BMI can affect mesothelin levels, which also often increase with age.
Identification of new candidate biomarkers
Due to an urgent need for better biomarkers for early detection of ovarian cancer and reliable biomarkers to monitor clinical response, ongoing efforts are focused on the application of state of the art technologies e.g. mass spectrometry and quantitative proteomic analysis to identify novel biomarkers [5]. These approaches however often generate multiple candidate biomarkers for further investigation, prioritization and clinical evaluation of which is an ongoing challenge. These methods allow comparison of multiplex biomarker panels and identification of novel differentially expressed proteins not previously linked to ovarian cancer.
Another powerful technology is microarray-based mRNA analysis which allows genome wide expression studies which have already enhanced the understanding of the genes and pathways which influence ovarian cancer progression, chemotherapy response and survival. For example, the candidate biomarkers osteopontin and kallikrein (Table 1) were discovered by this method.
Our own studies have revealed significant differences in the expression of fibroblast growth factor 1 (FGF1) and additional FGF pathway genes in ovarian cancers of different histologies (Fig. 2A) and in paired sensitive and resistant ovarian cancer cell lines (Fig. 2B). We have additionally shown that FGF1 expression is significantly inversely correlated with both progression-free (Fig. 2C) and overall survival in ovarian cancer patients [6]. We are therefore currently recruiting patients to longitudinal clinical studies to investigate whether FGF1 or additional related growth factors can predict disease progression and/or the development of treatment-limiting drug resistance.
MicroRNAs (miRNAs) are small non-coding RNAs (19–25 nucleotides) that regulate gene expression by binding to mRNA target sequences and disrupting translation [7]. MiRNAs have great potential as diagnostic and clinical response biomarkers in ovarian and additional cancers as miRNA expression can now routinely be quantitatively assessed in small biopsies and in formalin-fixed material. For example, approximately 30 miRNAs (including miR-21, miR-141, miR-203, miR-205 and miR-214) are differentially expressed in ovarian cancer [8], while miRNAs including miR-200a, miR-200b and miR-429 have also been associated with cancer recurrence and have been shown to predict survival. For example, high expression of miR-200, miR-141, miR-18a and low expression of let-7b, and miR-199a were found to predict poor survival in a cohort of 20 ovarian cancer patients [9]. Meanwhile, recent data from our own laboratory has identified multiple miRNAs including miR-125b and miR-130 associated with the development of platinum resistance. MiRNAs are particularly promising candidate biomarkers due to their stability, and abundant expression in solid cancers, whole blood and routinely collected plasma and serum samples.
Future directions
Due to the challenges of finding a single biomarker that can encompass the complexity and heterogeneity of ovarian cancer it is logical that optimization of a multi-biomarker panel may be the most practical approach, for example combining HE4 and mesothelin with CA125 to augment both sensitivity and specificity. This type of approach has recently been proposed in algorithms such as the Risk of Ovarian Malignancy Algorithm or ROMA which combines CA125 and HE4 levels with a sensitivity of 94% and specificity of 75%. [4]. Combinations of CA125 and mesothelin have also been found to detect more cancers than each biomarker alone. Several current studies have, however, suggested that combination biomarker analysis significantly increases the predictive power of CA125, but also unfortunately appears to decrease specificity. Ongoing studies therefore aim to develop improved biomarker panels suitable both for early detection and treatment guidance of ovarian cancer (Table 2). All of these results still require validation but they are indicative of the possible power of using a multi-biomarker panel in diagnostic tests and for monitoring the clinical responses of ovarian cancer.
Concluding remarks
An ideal biomarker for ovarian cancer will have a high enough sensitivity to correctly diagnose women with the disease and be specific enough to avoid false positive results. With ongoing efforts to identify biomarkers which match this ideal, hundreds of candidates with clinical relevance have been found but still require much validation before having a routine place in the clinic. It is expected that the future of ovarian cancer detection will be based on panels of combination serum-based biomarkers alongside biological imaging techniques to improve diagnosis, treatment and disease management.
References
1. Shapira I, Oswald M, Lovecchio J, Khalili H, Menzin A, Whyte J, Dos Santos L, Liang S, Bhuiya T, Keogh M, Mason C, Sultan K, Budman D, Gregersen PK, Lee AT. Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes. Br J Cancer 2014; 110: 976–983.
2. Nguyen L, Cardenas-Goicoechea SJ, Gordon P, Curtin C, Momeni M, Chuang L, Fishman D. Biomarkers for early detection of ovarian cancer. Women’s Health 2013; 9: 171–185; quiz 186–187.
3. Sarojini S, Tamir A, Lim H, LI S, Zhang S, Goy A, Pecora A, Suh KS. Early detection biomarkers for ovarian cancer. J Oncol. 2012; 15.
4. Jordan SM, Bristow RE. Ovarian cancer biomarkers as diagnostic triage tests. Current Biomarker Findings 2013; 3: 35–42.
5. Zhang B, Barekati Z, Kohler C, Radpour R, Asadollahi R, Holzgreve W, Zhong XY. Proteomics and biomarkers for ovarian cancer diagnosis. Ann Clin Lab Sci. 2010; 40: 218–225.
6. Smith G, NG MT, Shepherd L, Herrington CS, Gourley C, Ferguson MJ, Wolf CR. Individuality in Fgf1 expression significantly influences platinum resistance and progression-free survival in ovarian cancer. Br J Cancer 2012; 107: 1327–1336.
7. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
8. Zhang B, Cai FF, Zhong XY. An overview of biomarkers for the ovarian cancer diagnosis. Eur J Obstet Gynecol Reprod Biol. 2011; 158: 119–123.
9. Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, Kim JW, Kim S. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008; 14: 2690–2695.
10. Yurkovetsky Z, Skates S, Lomakin A, Nolen B, Pulsipher T, Modugno F, Marks J, Godwin A, Gorelik E, Jacobs I, Menon U, LU K, Badgwell D, Bast RC, JR, Lokshin AE. Development of a multimarker assay for early detection of ovarian cancer. J Clin Oncol. 2010; 28: 2159–2166.
11. SU F, Lang J, Kumar A, NG C, Hsieh B, Suchard MA, Reddy ST, Farias-Eisner R. Validation of candidate serum ovarian cancer biomarkers for early detection. Biomark Insights 2007; 2: 369–375.
12. Zhang Z, YU Y, XU F, Berchuck A, Van Haaften-Day C, Havrilesky LJ, de Bruijn HW, van der Zee AG, Woolas RP, Jacobs IJ, Skates S, Chan DW, Bast RC, Jr. Combining multiple serum tumor markers improves detection of stage I epithelial ovarian cancer. Gynecol Oncol. 2007; 107: 526–531.
13. Gorelik E, Landsittel DP, Marrangoni AM, Modugno F, Velikokhatnaya L, Winans MT, Bigbee WL, Herberman RB, Lokshin AE. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2005; 14: 981–987.
14. Lokshin AE, Winans M, Landsittel D, Marrangoni AM, Velikokhatnaya L, Modugno F, Nolen BM, Gorelik E. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecol Oncol. 2006; 102: 244–251.
The authors
Emma L. Joseph1 BSc; Michelle J. Ferguson2 MBChB, MD; and Gillian Smith1* PhD
1Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee UK
2Tayside Cancer Centre, Ninewells Hospital & Medical School, Dundee UK
*Corresponding author
E-mail: g.smith@dundee.ac.uk
RX series – Precision. Reliability. Accuracy
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, /in Featured Articles /by 3wmediaThe Aptima HIV-1 Quand Dx assay
, /in Featured Articles /by 3wmediaClinical response biomarkers in ovarian cancer: current challenges and future directions
, /in Featured Articles /by 3wmediaEffective screening strategies have not yet been developed for the early detection of ovarian cancer. The serum biomarker CA125, routinely used to aid diagnosis and monitor treatment response, is not informative in all patients. Recent analytical developments have prioritized promising candidate novel biomarkers or multi-biomarker panels for future clinical evaluation.
by E. L. Joseph, Dr M. J. Ferguson and Dr G. Smith
Introduction to ovarian cancer
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and the fifth leading cause of cancer related death among women with around 140 000 annual deaths worldwide. EOC can develop as one of four histotypes with the serous histotype being the most common and most aggressive. The remaining three non-serous histotypes, endometrioid, clear cell and mucinous cancers present less frequently. High grade serous ovarian cancer, heterogeneous in nature and rapidly progressive, has a poor prognosis, where a major contributing factor is the lack of ability to diagnose the disease at a sufficiently early stage to facilitate curative surgery. The 5-year survival rate is less than 30% for patients presenting with advanced disease spread beyond the ovaries (FIGO Stage 3/4), but if detected earlier combination therapy of cytoreductive surgery and adjuvant or neo-adjuvant chemotherapy with platinum and taxane-based drugs has the potential to cure 90% of patients [1]. Consequently the identification of biomarkers capable of detecting ovarian cancer at the earliest stages and monitoring disease progression are inherently important in tackling this lethal disease. Due to its prevalence, the ideal biomarker for detecting early stage ovarian cancer requires an extremely high specificity (>99%) and a minimum sensitivity of 75% [2]. Despite extensive research, no optimal ovarian cancer biomarker has yet been identified and such high specificity is unlikely to be met by a single agent. Many promising candidate biomarkers are however currently undergoing evaluation in clinical trials.
CA125
The only ovarian cancer biomarker routinely used in the clinic is cancer antigen 125 (CA125; mucin 16) currently considered the ‘gold standard’ cancer biomarker despite its limitations. In the majority of patients with EOC, expression of the CA125 glycoprotein is raised above the normal reference range (>35 U/ml blood), but it only has a sensitivity of 50% to 60% with a specificity of 90% in early stage postmenopausal patients [3]. Several factors, however, limit the utility of CA125 in routine population screening: it is not expressed in 20% of ovarian cancers, is only significantly elevated in 47% of early stage ovarian cancers (although increasing to 80–90% in advanced stage cancers) and can be raised in many benign conditions including endometriosis and peritonitis. Variability in CA125 expression throughout the menstrual cycle and in pregnancy is also a common confounding issue. A major clinical utility of CA125, however, is related to its ability to commonly reflect clinical response following chemotherapy treatment and as such is often successfully used to monitor a patient’s progress through chemotherapy. A reduction in CA125 expression during treatment is considered a positive prognostic outcome for the patient and serial serum measurements are currently used to predict therapeutic outcomes and estimate stability of the disease (Fig. 1).
Biomarkers under evaluation
There have now been multiple attempts to identify novel ovarian cancer biomarkers with varying success. The most promising serum biomarkers include HE4 and mesothelin.
HE4
Human epididymis protein 4 (HE4) has been shown to be consistently elevated above the normal level (151 pM) in ovarian cancers, with sensitivity of 95% and specificity of 73%. HE4 is differentially expressed in specific subtypes of ovarian cancer, potentially allowing clinicians to distinguish histotypes to aid treatment; HE4 was found to be overexpressed in 100% of endometrioid cancers, 93% of serous cancers but only 50% of clear cell cancers [4]. Unlike CA125, it is less likely to produce false positives in benign masses and it has also been proposed to be the best candidate biomarker for early detection of Stage I disease despite sensitivity and specificity of 46% and 95% respectively [2]. HE4 has recently obtained FDA approval in the USA for monitoring recurrence or progression of EOC and, in comparative tests, has been found to be superior to CA125 in classifying benign and borderline ovarian cancers.
Mesothelin
Mesothelin is a glycoprotein expressed by mesothelial cells, the expression of which has been found to be raised in mesothelioma, pancreatic and ovarian cancers.
It can be easily measured in both urine and serum, highlighting its potential as a non-invasive biomarker. Serum mesothelin levels were found to be increased in approximately 60% of ovarian cancers with 98% specificity.
One study found elevation of mesothelin in 42% of urine assays as opposed to 12% serum assays of early stage EOCs at 95% specificity which reinforces the potential of this glycoprotein as an early detection biomarker and the use of urine in preference to serum [2]. Higher levels of mesothelin were also found to be associated with poorer overall survival in patients following optimal debulking surgery or who have advanced stage ovarian cancer. A recent study, however, revealed that lifestyle choices such as smoking and BMI can affect mesothelin levels, which also often increase with age.
Identification of new candidate biomarkers
Due to an urgent need for better biomarkers for early detection of ovarian cancer and reliable biomarkers to monitor clinical response, ongoing efforts are focused on the application of state of the art technologies e.g. mass spectrometry and quantitative proteomic analysis to identify novel biomarkers [5]. These approaches however often generate multiple candidate biomarkers for further investigation, prioritization and clinical evaluation of which is an ongoing challenge. These methods allow comparison of multiplex biomarker panels and identification of novel differentially expressed proteins not previously linked to ovarian cancer.
Another powerful technology is microarray-based mRNA analysis which allows genome wide expression studies which have already enhanced the understanding of the genes and pathways which influence ovarian cancer progression, chemotherapy response and survival. For example, the candidate biomarkers osteopontin and kallikrein (Table 1) were discovered by this method.
Our own studies have revealed significant differences in the expression of fibroblast growth factor 1 (FGF1) and additional FGF pathway genes in ovarian cancers of different histologies (Fig. 2A) and in paired sensitive and resistant ovarian cancer cell lines (Fig. 2B). We have additionally shown that FGF1 expression is significantly inversely correlated with both progression-free (Fig. 2C) and overall survival in ovarian cancer patients [6]. We are therefore currently recruiting patients to longitudinal clinical studies to investigate whether FGF1 or additional related growth factors can predict disease progression and/or the development of treatment-limiting drug resistance.
MicroRNAs (miRNAs) are small non-coding RNAs (19–25 nucleotides) that regulate gene expression by binding to mRNA target sequences and disrupting translation [7]. MiRNAs have great potential as diagnostic and clinical response biomarkers in ovarian and additional cancers as miRNA expression can now routinely be quantitatively assessed in small biopsies and in formalin-fixed material. For example, approximately 30 miRNAs (including miR-21, miR-141, miR-203, miR-205 and miR-214) are differentially expressed in ovarian cancer [8], while miRNAs including miR-200a, miR-200b and miR-429 have also been associated with cancer recurrence and have been shown to predict survival. For example, high expression of miR-200, miR-141, miR-18a and low expression of let-7b, and miR-199a were found to predict poor survival in a cohort of 20 ovarian cancer patients [9]. Meanwhile, recent data from our own laboratory has identified multiple miRNAs including miR-125b and miR-130 associated with the development of platinum resistance. MiRNAs are particularly promising candidate biomarkers due to their stability, and abundant expression in solid cancers, whole blood and routinely collected plasma and serum samples.
Future directions
Due to the challenges of finding a single biomarker that can encompass the complexity and heterogeneity of ovarian cancer it is logical that optimization of a multi-biomarker panel may be the most practical approach, for example combining HE4 and mesothelin with CA125 to augment both sensitivity and specificity. This type of approach has recently been proposed in algorithms such as the Risk of Ovarian Malignancy Algorithm or ROMA which combines CA125 and HE4 levels with a sensitivity of 94% and specificity of 75%. [4]. Combinations of CA125 and mesothelin have also been found to detect more cancers than each biomarker alone. Several current studies have, however, suggested that combination biomarker analysis significantly increases the predictive power of CA125, but also unfortunately appears to decrease specificity. Ongoing studies therefore aim to develop improved biomarker panels suitable both for early detection and treatment guidance of ovarian cancer (Table 2). All of these results still require validation but they are indicative of the possible power of using a multi-biomarker panel in diagnostic tests and for monitoring the clinical responses of ovarian cancer.
Concluding remarks
An ideal biomarker for ovarian cancer will have a high enough sensitivity to correctly diagnose women with the disease and be specific enough to avoid false positive results. With ongoing efforts to identify biomarkers which match this ideal, hundreds of candidates with clinical relevance have been found but still require much validation before having a routine place in the clinic. It is expected that the future of ovarian cancer detection will be based on panels of combination serum-based biomarkers alongside biological imaging techniques to improve diagnosis, treatment and disease management.
References
1. Shapira I, Oswald M, Lovecchio J, Khalili H, Menzin A, Whyte J, Dos Santos L, Liang S, Bhuiya T, Keogh M, Mason C, Sultan K, Budman D, Gregersen PK, Lee AT. Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes. Br J Cancer 2014; 110: 976–983.
2. Nguyen L, Cardenas-Goicoechea SJ, Gordon P, Curtin C, Momeni M, Chuang L, Fishman D. Biomarkers for early detection of ovarian cancer. Women’s Health 2013; 9: 171–185; quiz 186–187.
3. Sarojini S, Tamir A, Lim H, LI S, Zhang S, Goy A, Pecora A, Suh KS. Early detection biomarkers for ovarian cancer. J Oncol. 2012; 15.
4. Jordan SM, Bristow RE. Ovarian cancer biomarkers as diagnostic triage tests. Current Biomarker Findings 2013; 3: 35–42.
5. Zhang B, Barekati Z, Kohler C, Radpour R, Asadollahi R, Holzgreve W, Zhong XY. Proteomics and biomarkers for ovarian cancer diagnosis. Ann Clin Lab Sci. 2010; 40: 218–225.
6. Smith G, NG MT, Shepherd L, Herrington CS, Gourley C, Ferguson MJ, Wolf CR. Individuality in Fgf1 expression significantly influences platinum resistance and progression-free survival in ovarian cancer. Br J Cancer 2012; 107: 1327–1336.
7. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
8. Zhang B, Cai FF, Zhong XY. An overview of biomarkers for the ovarian cancer diagnosis. Eur J Obstet Gynecol Reprod Biol. 2011; 158: 119–123.
9. Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, Kim JW, Kim S. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008; 14: 2690–2695.
10. Yurkovetsky Z, Skates S, Lomakin A, Nolen B, Pulsipher T, Modugno F, Marks J, Godwin A, Gorelik E, Jacobs I, Menon U, LU K, Badgwell D, Bast RC, JR, Lokshin AE. Development of a multimarker assay for early detection of ovarian cancer. J Clin Oncol. 2010; 28: 2159–2166.
11. SU F, Lang J, Kumar A, NG C, Hsieh B, Suchard MA, Reddy ST, Farias-Eisner R. Validation of candidate serum ovarian cancer biomarkers for early detection. Biomark Insights 2007; 2: 369–375.
12. Zhang Z, YU Y, XU F, Berchuck A, Van Haaften-Day C, Havrilesky LJ, de Bruijn HW, van der Zee AG, Woolas RP, Jacobs IJ, Skates S, Chan DW, Bast RC, Jr. Combining multiple serum tumor markers improves detection of stage I epithelial ovarian cancer. Gynecol Oncol. 2007; 107: 526–531.
13. Gorelik E, Landsittel DP, Marrangoni AM, Modugno F, Velikokhatnaya L, Winans MT, Bigbee WL, Herberman RB, Lokshin AE. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2005; 14: 981–987.
14. Lokshin AE, Winans M, Landsittel D, Marrangoni AM, Velikokhatnaya L, Modugno F, Nolen BM, Gorelik E. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecol Oncol. 2006; 102: 244–251.
The authors
Emma L. Joseph1 BSc; Michelle J. Ferguson2 MBChB, MD; and Gillian Smith1* PhD
1Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee UK
2Tayside Cancer Centre, Ninewells Hospital & Medical School, Dundee UK
*Corresponding author
E-mail: g.smith@dundee.ac.uk
Unanswered questions about testing for low testosterone in men
, /in Featured Articles /by 3wmediaTestosterone is a steroid hormone that develops and maintains the primary and secondary sex characteristics in men. In recent years, there has been an explosive increase in prescriptions for testosterone replacement therapy (TRT) in adult men who are thought to have adult-onset hyopgonadism. This increase has been fueled by changing demographics and by increased public awareness of so-called “low-T” syndrome. Despite recent controversies about risks for cardiovascular complications in men receiving TRT, the trend of increased testing and treatment for low-T is likely to continue. This article explores current controversies and unanswered questions regarding testing for low-T in men. Topics covered include variations in reference intervals for testosterone and thresholds for interpretation of results. Controversies and questions surrounding testing for free (unbound) testosterone will also be explored. Finally, the emerging evidence regarding the roles of dihydrotestosterone and estradiol will be discussed in the context of testing for low-T.
by Dr Michael Samoszuk
Testosterone metabolites include estradiol (produced by the aromatase enzyme found in fat and other tissues such as testes) and dihydrotestosterone (DHT)-an androgenic hormone that is approximately three- to ten-times more potent than testosterone. DHT is produced from testosterone by 5-alpha reductase, an enzyme that is found primarily in hair follicles, prostate, testes, and adrenal glands but not in skeletal muscle.
In recent years, there has been an explosive increase in prescriptions (Figure 1) for testosterone replacement therapy (TRT) in men who are thought to have adult onset hypo-gonadism, a condition that is often referred to as “low-T”. This condition is characterized by a variety of signs and symptoms, including loss of body hair, accumulation of visceral and body fat, loss of skeletal muscle, anemia, mood disturbances, loss of libido, and erectile dysfunction. Because of changing demographics and increased awareness of low-T due to marketing campaigns, it is likely that testing for (and treatment of) low-T will continue to increase significantly in the next five years. This increased testing and treatment for low-T will probably occur despite recent controversies about the cardiovascular risks that may be associated with TRT.
How should total testosterone levels be interpreted in men being tested for low-T?
There is considerable variation in the reference intervals for total testosterone assays that are produced by various manufacturers of in vitro diagnostics (Table 1). It is notable that the reference intervals are based on the range of values between the 5th-95th percentiles of men of various ages. Because the populations of men that were used to derive these reference intervals are poorly defined with respect to age distribution and possible symptoms of low-T, it is unclear if these reference intervals provide a reliable basis for interpreting test results from men being tested for low-T. Of particular concern are the lower limits of the reference intervals, which may be too low to identify the significant proportion of men who are truly hypo-gonadal but whose total testosterone levels fall above the 5th percentile of the reference range.
Reference intervals for total testosterone levels reported by reference laboratories also have considerable variation (Table 2). The variation is of particular concern at the low end of the reference interval because many clinicians use this value to determine whether or not a man should be diagnosed as having low-T. Unanswered questions regarding the use of reference intervals that are reported by clinical laboratories include:
An emerging way to interpret total testosterone levels in men is to use a clinical threshold (cut-off) value for the level. Although there are significant differences in the definitions of the clinical thresholds (Table 3), it appears that the clinical threshold for diagnosing low-T probably lies somewhere between 300-500 ng/dL. Notably, this range lies considerably above the 5th percentiles for testosterone values that are listed in Tables 1 and 2. It should also be noted that all sources of the clinical thresholds listed in Table 3 also recommend the primacy of clinical signs and symptoms when interpreting total testosterone values.
Unanswered questions regarding the use of clinical threshold values are:
Should free testosterone be measured?
Testosterone circulates in the blood in a free (unbound) form and a bound form. Sex-hormone binding globulin (SHBG) and albumin are the primary sources of binding of testosterone.
Approximately 2% of total testosterone circulates in the free form. Current thinking is that the free testosterone is mostly responsible for the biological activity of the hormone, and the bound form is thought to be mostly inactive. The free and bound forms can be directly measured by a variety of methods, or a mathematical formula can be used to calculate the percentage of free testosterone, based on the values for total testosterone, SHBG and albumin.
There is substantial confusion over the best way to determine free testosterone and how to interpret the results. Unanswered questions include:
Is there a role for testing for DHT?
Testing for DHT is not commonly performed in the evaluation of men being evaluated or treated for hypo-gonadism. At this time, it is not clear how to interpret the test results. There is some evidence, however, that treatment of low-T with topical testosterone preparations can sometimes preferentially elevate the DHT levels due to the presence of 5-alpha reductase in skin and hair follicles. In theory, this phenomenon could account for so-called treatment failures of men who receive topical therapy. It is likely that as our understanding of TRT improves, there will be an increased interest in clinical testing for this metabolite of testosterone.
Is it necessary or helpful to measure estradiol in men being evaluated or treated for low testosterone?
There is now considerable evidence that estradiol levels in men play an important role in modulating the effects of testosterone on sexual function, body fat, lean muscle mass, and bone density. Nevertheless, estradiol levels are still not commonly measured in men who are being evaluated or treated for low-T. This is because the interpretive criteria for such testing are still not well understood. In some men, estradiol levels are measured in order to determine if TRT is causing an increase in estradiol due to aromatization of the testosterone. This can lead to symptoms of high estradiol such as bloating, fluid retention, and breast tenderness. Some experts now recommend calculating a ratio of testosterone to estradiol, but this approach is not yet widely accepted. Nevertheless, it is likely that clinical laboratories will experience an increased demand for estradiol testing in men as our understanding and prevalence of TRT increase.
Conclusion
From the preceding discussion, it should be apparent that our understanding of laboratory testing for low-T in men lags considerably behind the growing demand for testing and treatment of low-T. Clinical laboratories, manufacturers of in vitro diagnostic tests, and clinicians should be aware of the many unanswered questions in this field. They should also begin to prepare to educate themselves about the important changes in this field that are likely to occur in the next few years. For further details about this subject, the interested reader is referred to the following sources.
References
Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM. Testosterone therapy in adult men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2006: 91, 1995-2010.
Finkelstein JS, et al. Gonadal steroids and body composition, strength and sexual function in men. N Engl J Med 2013: 369, 1011-22.
Morgentaler A, Khera M, Maggi M, Zitzmann M. Commentary: Who is a candidate for testosterone therapy? A synthesis of international expert opinions. J Sex Med 2014: 11, 2636-45.
www.peaktestosterone.com A testosterone and men’s health blog that is an excellent source of information and peer-reviewed publications on this topic.
The author
Michael Samoszuk, M.D.
Chief Medical Officer
Beckman Coulter Diagnostics
Sekisui Diagnostics – Your global partner for over 30 years
, /in Featured Articles /by 3wmediaSekisui Diagnostics is a global diagnostics manufacturer focused on the clinical chemistry, coagulation, infectious disease, and enzyme markets. Headquartered in Lexington, MA, we have 10 facilities in 6 countries and over 500 employees worldwide. We are dedicated to delivering differentiated products, instrument systems, and services that support the improvement of patient care worldwide.
Formerly Genzyme Diagnostics, we changed our name in 2011 when we began a fresh chapter as part of the Sekisui Medical family. We remain focused on innovation and are now supported by the resources of our global parent corporation. We are a diverse company with broad product lines, a global sales and distribution network, extensive product development capabilities, state of the art manufacturing facilities, and deep diagnostics expertise.
Our core competency is to work with healthcare professionals and diagnostic manufacturers to deliver high-quality diagnostic products that help improve patient health.
Today’s healthcare professionals have a challenging job. They need to deliver quality, cost-effective test results that support patient care and facilitate effective outcomes. Accurate, reliable diagnostic tests and systems from Sekisui Diagnostics can help provide the quality results doctors, patients, and researchers expect in a fast, cost-effective, and responsible way.
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Carbapenemases: a major threat to healthcare
, /in Featured Articles /by 3wmediaBacterial resistance to antibiotics is a major health and economic problem recognized today by national and international policy makers. The Enterobacteriaceae belong to the commensal human gut flora and are frequently the cause of community- and healthcare-associated infections (HAI). Infections with Klebsiella pneumoniae are usually hospital-acquired and occur primarily in patients with impaired host defences while Escherichia coli are mostly involved in urinary tract infections. Acinetobacter and Pseudomonas species are opportunistic pathogens frequently isolated from healthcare settings where they cause life-threatening infections particularly in immunocompromised patients.
For several years, in Europe and worldwide, Enterobacteriaceae, mainly K. pneumoniae and E. coli but also non-fermenting bacteria (Acinetobacter baumannii and ) have become resistant to the main antibiotics, i.e. ß-lactam antibiotics, fluoroquinolones and aminoglycosides [1]. In particular, the US “Center for Disease Control and Prevention” (CDC) pointed the carbapenemase-producing Enterobacteriaceae (CPE) (or Carbapenem Resistant Enterobacteriaceae, CRE) among the three microorganisms exhibiting the most urgent health risk. Carbapenemases are indeed enzymes that inactivate ß-lactam antibiotics like carbapenems that currently constitute the last resort for treating multi-drugs resistant Gram-negative bacteria. Moreover, production of carbapenemases in these bacteria is most often associated with the expression of additional resistance mechanisms to other classes of antibiotics such as aminoglycosides, fluoroquinolones and cotrimoxazole, leading to bacteria resistant to all available antibiotics, so-called pan-resistant bacteria [2].
Carbapenemases resistance
Carbapenems are a class of broad-spectrum ß-lactam antibiotics with very broad activity and have therefore become the empirical treatment of choice in countries where infections due to Extended-Spectrum ß-Lactamases-producing bacteria are common. Resistance to carbapenems in Enterobacteriaceae is linked to either decreased permeability because of porine alteration or loss combined with production of a ß-lactamase with poor carbapenemase activity or, more worryingly, to the enzymatic breakdown of the antibiotic by a carbapenem-hydrolyzing ß-lactamase [3].These so called carbapenemases can hydrolyse and hence deactivate several kinds of antibiotics ranging from penicillins to cephalosporins, monobactams and carbapenems.
The most frequent carbapenemases in Enterobacteriaceae reported in Europe belong to three molecular classes according to the Ambler classification:
– class A carbapenemases hydrolyse almost all ß-lactams including carbapenems. Klebsiella pneumoniae carbapenemase (KPC) are the most frequent enzymes of this group that show a very high spreading capability [4] . KPC, contrary to other class A ß-lactamase, is inhibited by boronic acid and its derivatives.
– class B carbapenemases, belonging to metallo-ß-lactamases, including Verona integron-encoded metallo-ß-lactamase (VIM), IMP and the New Delhi metallo-ß-lactamase-1 (NDM-1) can hydrolyse all ß-lactams but monobactams. These enzymes are inhibited in vitro by EDTA and dipicolinic acid that are used in confirmatory tests for the presence of metallo ß-lactamases (MBL). NDM is the most frequent MBL present in Enterobacteriaceae. Originally detected in the Asian subcontinent, NDM is now spreading worldwide and causing outbreaks in Western countries.
– class D carbapenemases including the OXA carbapenem-hydrolysing oxacillinases hydrolyse penicillins but show lower activity against carbapenems, and no activity against extended-spectrum cephalosporins [5)]. OXA-48 is the main enzyme of this family and is now spreading worldwide even in the community although only a few cases are reported in the USA. OXA-48 hydrolyses all penicillins including temocillin. The resistance to temocillin primarly evidenced by a Belgian group [6] is now recommended as a marker of the possible presence of OXA-48. Resistance to carbapenems in OXA-48-producing CPE is variable with minimal inhibitory concentration againt carbapenems varying between less than 0.5 µg/mL to more than 256 µg/mL. This mechanism is very difficult to detect and no confirmatory test currently exists as OXA-48 is not specifically inhibited by clavulanic acid, boronic acid or EDTA. To date, only expensive molecular tests are able to confirm the presence of OXA-48. The rapid and global expansion of CPE is a threat to healthcare and patient safety worldwide, as it seriously curtails the ability to cure infections. Infections due to CPE are associated with higher in-hospital morbidity and mortality [7].
Carbapenemases epidemiology
According to the report summarizing the results from 39 European countries [8], six levels of occurrence of CPE have been defined i.e. endemic situation (level 1), inter-regional spread (level 2), regional spread (level 3), sporadic hospital outbreaks (level 4), single hospital outbreaks (level 5) and sporadic occurrence (level 6).
Nevertheless, specific occurrence may also vary depending on the type of CPE.
Discussion
Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat to healthcare and are frequently resistant to many other antibiotics besides carbapenems leaving few treatment options [9, 10]. Rapid diagnostic tests that can be performed directly on clinical specimens or blood cultures are urgently needed in order to save an average of 24 hours compared to the results obtained by culture. Some commercial tests of this type targeting carbapenemases from CPE currently exist and are based either on molecular amplification of specific genes associated with resistance or on molecular hybridization.
Some tests are able to target all carbapenemases of clinical interest, or other resistance mechanisms alongside with accurate species identification.
However, these tests require specific equipment and are extremely expensive (80 Euros being an average price).
Multiplex real-time PCR tests allow the detection of the genes encoding for the main carbapenemases directly from samples or feces but do not detect all variants of the genes of interest in a single operation.
Other molecular biology tests can be performed on isolated colonies from culture. These tests are costly (average cost of 40 €), quite labour-intensive and often do not deliver results before the next morning, when the susceptibility testing is already available. Molecular biology tests only partially meet the needs in carbapenemase identification. Either these tests do not cover the complete range of targets or cannot distinguish between different carbapenemases and, in any case, do not give information on the level of gene expression and thus the level of bacterial resistance. The high price of these techniques and/or the need for expensive equipment, dedicated areas and specially trained personnel restrict their use to a limited number of specialized laboratories.
Rapid phenotypic tests directly performed on bacterial colonies and based on the hydrolysis of a carbapenem with colorimetric shift are now available at a reasonable price. MALDI-TOF mass spectrometry is also proposed, however, this technology requires the use of expensive equipment together with specific software analysis. All the above phenotypic tests only partially meet the needs of clinical laboratories. On the one hand, most of them require the use of antibiotics with stability problems, and secondly, the time for obtaining a result with these tests is not totally satisfactory in terms of integration into the laboratory workflow that would ensure results in a short time and allow quick decision for optimal impact. On the other hand these phenotypic tests do not identify the exact type of carbapenemase and ideally require subsequent procedures using a molecular method to achieve identification. Among CPE, OXA-48 represents the most challenging resistance mechanism to be identified that would need a rapid and easy to use test to be performed in routine labs.
References
1. Carbapenemase-producing bacteria in Europe. Interim results from the European Survey on carbapenemase-producing Enterobacteriaceae (EuSCAPE) project 2013.
2. Souli M, Galani I, and Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveillance 2008; 13(47).
3. Nordmann P, Naas T, and Poirel L. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
4. Naas T, Cuzon G, Villegas M-V, Lartigue M-F, Quinn JP, and Nordmann P. Genetic structures at the origin of acquisition of the ß-lactamase blaKPC gene Antimicrobial Agents and Chemotherapy 2008; 52(4): 1257–1263.
5. Nordmann P, Naas T, and Poirel P. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
6. Glupczynski Y, Huang TD, Bouchahrouf W, Rezende de Castro R, Bauraing C, Gérard M, Verbruggen AM, Deplano A, Denis O, Bogaerts P. Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals. Int J Antimicrob Agents 2012; 39(2):168-72.
7. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, et al. Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 2009, 30:972–6.
8. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013, Euro Surveill. 2013;18(28):pii=20525.
9. Cantón R, Akóva M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among in Europe, Clin Microbiol Infect. 2012;18(5):413–31.
10. Hawkey PM. The growing burden of antimicrobial resistance, J Antimicrob Chemother. 2008;62(Suppl 1):i1–i9.
The authors
Isabelle OTE, Laetitia AVRAIN, Pascal MERTENS, Thierry LECLIPTEUX
R&D Department, Coris BioConcept,
Parc Scientifique Crealys,
29A, rue Jean Sonet,
B-5032 Gembloux, Belgium