Clinical usefulness of circulating tumor markers

Introduction

Classically, the term tumor marker (TM) refers to substances directly produced by cancer cells or by other cells in response to a tumor. Circulating TMs are present in the blood, and other body fluids, like urine and pleural or peritoneal effusions. Table 1 summarizes the main TMs used in clinical practice, showing the associated malignancies. The clinical role of circulating TMs has remained invariable over the years and their usefulness controversial, in spite of their wide potential application in cancer diagnosis, prognosis and monitoring (Table 2).

TMs are cancer related substances but they are not specific, as they can be also expressed by healthy tissues, and small concentrations could be detected in healthy subject’s bloodstream. Cancer patients may have raised levels of TMs, which are correlated with the disease stage. However, false positive results could be found in patients with benign conditions, including processes that increase their release or reduce their catabolism. Increased plasma concentrations of TMs could lead to unnecessary tests to confirm or rule out the suspected neoplasm and also psychological impact on patients. In order to avoid these undesirable effects, it is essential to know the potential causes of false positives. On this point, the Clinical Chemistry and Laboratory Medicine (CCLM) journal, through several publications, has facilitate the knowledge on physiopathological processes that increase the concentrations of TMs [1].

This review analyzes the usefulness of most used TMs in frequent epithelial tumors, based on recent evidence and clinical guidelines.

The role of tumor markers according to clinical guidelines

In recent years, there has been a notable increase in requests for circulating TMs, mostly due to inadequate use of their measurement [2]. In a large study published in CCLM, Moreno-Campoy et al. [3] indicate that only 39.88% requests out of 23,059 in 5,080 patients with neoplastic diseases have been classified as adequate according to current clinical guidelines. In fact, assessing the adequacy of TMs according to their clinical usefulness remains the main challenge.

Based on available scientific evidence, clinical practice guidelines should be used to define, in which cases TMs are clinically valid and, therefore, offer an improvement in clinical results and the patient’s quality of life, also contributing to maintain an adequate financial balance.

However, clinical guidelines do not always agree and, occasionally their recommendations can be controversial. The extensive tumor-by-tumor review on available guidelines carried out by Gion et al. [4], [5], [6], represents a valuable contribution. With the Appraisal of Guidelines for Research & Evaluation (AGREE II) tool, this review provides assessment of each guide to facilitate quality comparison.

These guidelines, despite their overall quality rating is high, do not always consider sufficiently the pre-analytical, analytical and post-analytical variables that are involved in the measurement of TMs. Among the guidelines that do take this into account is the National Academy of Clinical Biochemistry (NACB), which, despite not having been updated since 2008, presents a broad description of the laboratory variables that can affect the TMs measurement [7, 8]. All the variables that affect the analytical process, from pre-analytical conditions to methodological differences, have a great impact on the results and their correct interpretation. Not taking these variables into account can cause significant errors in the clinical allocation of TMs. This topic has been extensively reviewed by the CCLM journal, as reflected in the following publications.

Filella et al. [9] remarked that the quality of prostate cancer (PCa) early detection guidelines could be improved properly considering the laboratory issues in their development and proposed a list of questions that should be considered regarding PSA measurement. The authors highlight the lack of interchangeability between the different PSA assays, which remains unsolved despite the introduction of international WHO standards [10, 11]. This is an aspect that needs to be emphasized for all TMs and that sometimes, as happens with CA 19-9 [12], leads to notable inter-assay differences that can cause errors in their interpretation of results when they are not considered.

Additionally, among laboratory variables not generally considered in clinical guidelines, there are differences in measured TMs concentrations attributable to the use of different reagent lots overtime. These differences can potentially cause result misinterpretation. To minimize this risk, Solsvik et al. [13] recently proposed a simplified and pragmatic lot-to-lot evaluation processing in which information about multiple lot changes from different clinical laboratories can be accumulated nationally (Table 3).

Clinical value of tumor markers in cancer detection

Diagnostic confirmation and histological classification of cancer may be challenging. Delay in the diagnostic process compromise patient’s therapeutic options and consequently their prognosis. The ideal clinical usefulness of TMs is to facilitate, in a non-invasive, simple and fast way, cancer early diagnosis. However, most TMs lack sensitivity to detect tumors in early stages and therefore guidelines do not recommend them as a screening tool in asymptomatic patients. As a maximum, in patients with suggestive signs or symptoms of malignancy, the detection of elevated levels would support clinical suspicion. With few exceptions, such as α fetoprotein and human chorionic gonadotrophin in germ cell tumors [21] and prostate specific antigen (PSA) in PCa, most recommendations are against their use for cancer detection. Furthermore, TMs quantification may be useful in patients with cancer of unknown primary site to suggest the origin, reducing the hospitalization time, morbidity, and the number of tests for diagnosis [22].

The role of PSA has been relevant in PCa detection, although its use has long been highly controversial [23]. PSA low specificity, the risk of overdiagnosis, overtreatment on screening, along with treatment adverse effects and a questionable decrease in mortality caused by PCa are reasons against the use of PSA on screening [24].

A recent initiative promoted by the European Association of Urology (EAU) proposes to overcome the disjunctive between screening everyone and not screening anyone, choosing to use PSA in a more effective way [25–27]. This new strategy is based on the PSA ability for predict future diagnoses of PCa shown in different studies [14–20] (Table 2). A recent study published by Kovac et al. [20], analyzing the data from 10,968 individuals enrolled in the American PLCO screening trial, remarked the strong relationship between basal PSA concentration among patients aged 55–60 and the long-term diagnosis of clinically significant PCa.

EAU initiative considers that a personalized strategy can reduce the harms effects of screening, maintaining the reduction of metastases and death, according the European Randomized Study of Screening for Prostate Cancer [28]. The algorithm proposed by EAU is applied to well-informed subjects and starts with a baseline PSA that will determine the periodicity of subsequent measurements. For subjects with a baseline PSA lower than 1 μg/L, PSA should be measured after 5 years in individuals aged 50–59, whereas PSA measurements should be stopped for subjects aged 60–70. On the other hand, PSA measurement should be done after 2–4 years when the baseline PSA is between 1 and 3 μg/L. Finally, risk stratification based on risk calculators and magnetic resonance imaging to select men for prostate biopsy is proposed when PSA is higher than 3 μg/L. Recently, the European Commission recommended the implementation of a PCa screening program based on PSA testing for men up to 70, in combination with additional magnetic resonance imaging as a follow-up test [29, 30].

One example of recommendations against the use of TM in the diagnosis setting is breast cancer (BC), the neoplasm with the highest incidence and mortality in women. In this case, a large number of circulating biomarkers have been studied, including mucins (carbohydrate antigen 15.3 [CA 15.3], cancer antigen 27–29 [CA 27–29]), oncoproteins (serum HER2 [sHER2]), oncofoetal proteins (carcinoembryonic antigen [CEA]), and cytokeratins (tissue polypeptide antigen [TPA], tissue polypeptide specific antigen [TPS] and cytokeratin-19 fragment [CYFRA 21-1]) [31]. Among all biomarkers, CA 15.3 is the most valuable; however, its sensitivity is conditioned by the cancer stage. In a prospective study including 2,062 patients with untreated primary BC, the overall sensitivity of CA 15.3 was higher than CEA (19.6 vs. 12.7%); and the combined assessment, increased sensitivity to 28% [32]. In spite of less evidence on the use of CEA, the combination of both markers provides complementary information and increases performance [33, 34]. However, due to low sensitivity, their measurement is not recommended for screening or early diagnosis, as reflected unanimously in international clinical guidelines [34], [35], [36], [37]. Given their relationship with disease extension, it is reasonable to think that they can be useful complementing patient staging [38]. Nevertheless, the European Group on Tumor Markers (EGTM) is the only one that supports its value for prognosis and staging, as high levels of both TMs in patients with localized disease might facilitate the detection of subclinical metastases [33].

Regarding lung cancer (LC), specific biomarkers have not been yet identified and current TMs usefulness is still unclear. Despite improvements in diagnosis approaches and novel treatments (targeted therapies and immunotherapy), LC remains the leading cause of cancer mortality worldwide. Most patients do not exhibit specific symptoms on diagnosis and one in two patients is diagnosed with advanced or locally advanced stages, thus there is an urgent need to improve tools for early diagnosis. Current strategies for LC screening are based on low-dose computed tomography (LDCT), which is supported by positive results from The National Lung Cancer Screening Trial (NLST) and the European NELSON trial [39, 40]. However, LC screening is still far from global implementation and there are concerns regarding false positives, the risk of overdiagnosis, and differences in patient selection criteria.

Circulating biomarkers would optimize imaging screening in two ways: (1) as a pre-test to refine risk stratification in combination with current selection criteria (age and tobacco exposure), and (2) as a post-test to help clinical decision-making in the management of indeterminate lung nodules (ILNs), also reducing unnecessary LDCT follow-ups [41]. Different panels of biomarkers have been evaluated but to date, none has shown sufficient sensitivity and specificity to be implemented for screening purposes. As an example, a panel of three TMs (CEA, cancer antigen 125 [CA 125], CYFRA 21-1) and 1 autoantibody (AAb) showed 71% sensitivity and 88% specificity in a selected high-risk cohort (based on age and smoking history as risk factors) [42]. However clinical validation in an independent cohort demonstrated lower sensitivity (49%) [43]. A recent study, analyzed a multi-analyte blood test to detect several cancers at early-stages. This test includes the TMs CA 125, CEA and CA 19-9; among other proteins and mutations in cell-free DNA (cfDNA). Although combining conventional TMs with novel circulating biomarkers seems a promising strategy for other cancers, the sensitivity obtained for LC was the lowest (39%) [44]. In conclusion, neither existing circulating biomarkers nor a combination of them is recommended in asymptomatic populations or specific high-risk groups.

Interestingly, circulating TMs have potential for helping diagnosis in patients with suggestive symptoms of LC (ILN, hemoptysis, dyspnea, etc.). According to the NACB, the most useful TMs aiding LC detection are: CEA, CYFRA 21-1, neuron specific enolase (NSE), squamous cell carcinoma antigen (SCC), and pro-gastrin releasing peptide (ProGRP) [45].

Additionally, the detection of ILN is very common; approximately 1.5 million nodules every year in the United States. Although the majority are benign, the effective detection and treatment of malignant nodules are crucial to reduce mortality [39]. The prospective study including 3,144 patients with clinical suspicion of LC (33% with nodules), evaluates the performance of six combined TMs obtaining an 88.5% sensitivity and 82% specificity. The inclusion of TMs results increased the detection capacity (AUC from 0.85 to 0.93) of the conventional prediction model based on nodule size, age, and smoking status [46]. Thus, the most effective strategy to improve diagnostic performance is combining multiple biomarkers with image and clinical parameters.

TMs can also aid LC diagnosis in the histological classification of tumors, as the therapeutic conduct and prognosis differ depending on it. Different patterns can discriminate between non small cell lung carcinomas (NSCLC) and small cell lung carcinomas (SCLC), also suggesting the most probable histology: increased CEA in adenocarcinoma; CYFRA 21-1 and SCC in squamous cell carcinoma; and NSE and ProGRP in small cell lung cancer [45]. Despite evidence, no clinical guideline recommends the use of TMs as a tool for diagnostic or histological discrimination. Initial evaluation of patients with suspected LC includes general laboratory tests such as hematology, coagulation, and biochemical profile, without specifying the possibility of using TMs [47]. Only the NACB and the EGTM pointed out that TMs have considerable potential for differential diagnosis and histological subtyping, particularly in tumors of unknown origin [45, 48]. The British Thoracic Society guidelines for the investigation and management of pulmonary nodules reported that although some biomarkers show interesting results, further studies are required to validate their performance prospectively prior to be recommended for clinical practice [49].

The implementation of new circulating biomarkers for early diagnosis of epithelial ovarian cancer (EOC) is an unmet medical need. This cancer is considered as “silent killer” because it is the most deathly gynecologic malignancy, frequently diagnosed in advanced stages. Currently, CA 125 is the predominant TM in EOC, although it is not exempt from limitations, including low sensitivity and specificity. Despite the enormous efforts to discover new biomarkers, the only one that has reached an effective clinical implementation is HE4, authorized by the FDA in 2008 [50]. Many publications have shown HE4 as the most promising biomarker. Moore et al. demonstrated that HE4 has high sensitivity and specificity as well as greater sensitivity than CA 125 in early stages [51]. HE4 is an example of how a new biomarker becomes a clinical reality, but its clinical value has been less assessed than CA 125; some aspects remain unclear and still need further study, as pointed out in a recent meta-analysis [50].

Although the use of these TMs for screening purposes is generally not recommended, the measurement of CA 125 and transvaginal ultrasonography are reasonable options for women at high risk of ovarian cancer (e.g. patients with suggestive symptoms and carriers of germline pathogenic variants in BRCA1/2 genes), according to the American College of Obstetricians and Gynecologists (ACOG) and The National Institute for Health and Clinical Excellence (NICE) current guidelines [52–54]. This strategy is based on the fact that positive predictive value is low in general population due to the low incidence of EOC, which can result in a considerable number of false positives; but diagnostic efficacy could be improved if we apply TMs in high-risk groups.

In this scenario, the Risk of Ovarian Malignancy Algorithm (ROMA) which combines CA 125 and HE4 along with menopause status, has been proposed to estimate the ovarian cancer risk in patients with adnexal masses [55]. The aim is to identify high-risk patients to be referred to a gynecologic oncologist for further testing as well as reduce the number of unnecessary surgeries. In an exhaustive meta-analysis, HE4 measurement seems to be superior to CA 125 in terms of diagnostic performance for the identification of EOC in women with suspected gynecological disease [56]. Molina et al. concluded that HE4 is the TM of choice in EOC, with higher efficiency than CA 125 and ROMA algorithm. Moreover, ROMA may be used in those patients with HE4 negative and CA 125 positive results, increasing the TM utility in the diagnosis of pelvic masses [57]. But results are inconclusive, in a recent comparison, the performance of CA 125, HE4, ROMA and Copenhagen index (CPH-I) to preoperatively identify EOC or metastatic cancer in the ovary was evaluated. ROMA and CPH-I perform better than TMs alone to identify patients harboring EOC or metastasic cancer [58].

Colorectal cancer (CRC), the third most common cancer, accounts for approximately 1.9 million new cases and 0.9 million deaths per year globally. Even though CEA is the most studied TM, lack of sensitivity in early stages combined with the low prevalence of CRC in asymptomatic populations excludes its use for screening. Although CEA concentration is not sufficient for CRC diagnosis in the absence of confirmatory biopsy, it should be evaluated before surgery, as baseline levels add prognosis information. Preoperative serum CEA concentration has been described as an independent predictor of overall survival across all stages [59], and postoperative increased levels also suggest a worse outcome [60].

Role of tumor markers in the follow-up of patients with cancer

In general, clinical practice guidelines agree that the main clinical application of TM is during the follow-up of cancer patients. In this sense, the role of PSA in the management of PCa patients is well-established [61, 62]. In the management of advanced cancer patients, two scenarios must be considered: (1) the early detection of relapse after primary treatment and (2) monitoring therapy response in advanced disease.

In BC, the usefulness of TMs in disease follow-up is also controversial. Most recommendations from oncology scientific societies are against measuring CA 15.3 or CEA during post-operative surveillance [63–65]. This is based on lack of evidence that early detection of recurrence by TMs, even months before radiological or clinical findings, has a clinical benefit. TMs diagnostic capacity depends on recurrence location, being low for local but higher for metastatic recurrences [66]. In this line, the EGTM recommends serial measurement of CA 15.3 and CEA for early detection of recurrence or metastatic disease in patients with BC and no evidence of disease, if it would alter clinical management [33]. In the current scenario, where more effective therapeutic options are available for metastatic patients and TMs continue to be requested it would be interesting to prospectively evaluate whether early detection of recurrences has an impact on patient outcomes. The recent consensus from the Spanish societies of laboratory medicine and medical oncology (SEQC-SEOM), suggests that their measurement should be limited to patients with a high risk of recurrence [34].

Conversely, the usefulness of CA 15.3 and CEA in the follow-up of advanced disease is supported, with different nuances, by most scientific societies [34]. It is important to keep in mind that TMs results must be integrated with imaging tests and other relevant clinical information. Only in some situations, therapeutic decisions should be guided by TMs, for example when it is not possible to measure the degree of disease extension (e.g. non-measurable bone metastasis).

Concerning EOC, CA 125 is the most studied biomarker and its utility in follow-up is well known. The measurement of serum CA 125 during chemotherapy has long been used to evaluate treatment response, complementing imaging and clinical assessment. International clinical guidelines agree on its usefulness; however, there are different criteria to consider significant changes along the follow-up. Regarding the detection of recurrences, it has been suggested that HE4 outperforms CA 125 [67], but recent studies indicate that both TMs have a similar performance and, although HE4 adds information in some cases, CA 125 is the most reliable marker for disease monitoring [50].

Additionally, TMs have an important role in the non-invasive assessment of disease extension. Estimation of tumor burden is basic to select the best primary treatment for advanced EOC patients, but it is often challenging and usually performed through surgical procedures. As a recent study showed, both CA 125 and HE4 correlate to whole-body tumor burden assessed by PET/CT before the primary treatment, and HE4 has superior performance than CA 125 estimating peritoneal disease in patients with high-grade advanced OEC [68].

CEA is the TM of the highest value in the follow-up of patients with CRC, also strongly recommended by international guidelines. Different meta-analyses have shown that intensive follow-up including CEA monitoring is associated with significantly earlier detection of recurrence and has a significant impact on survival [69]. Thus, CEA monitoring is generally recommended after surgery, although there are discrepancies between different guidelines regarding the frequency of CEA testing [70–72]. Moreover, there is no definition of what constitutes a clinically significant increase in CEA concentration. Generally, differences of 25–30% between two values are considered significant, based on reference change values (RCV) [73]. CEA is also the marker of choice for monitoring treatment response and early detection of progression in patients with metastatic disease. Persistently increasing concentrations above baseline suggest progressive disease even in the absence of corroborating radiographs [74]. Thus, it is crucial to correctly interpret and confirm any increase, with special caution during the first 4–6 weeks of treatment, because transient elevations in absence of progression can occur.

Circulating biomarkers for precision oncology

Precision oncology seeks to achieve a more personalized and effective treatment of patients according to tumor characteristics. The improvements in molecular biology techniques and the identification of key driver genes have allowed the scientific community to go one step further in the knowledge of cancer molecular bases. This scenario has facilitated the approval of targeted therapies that are more effective and have demonstrated better patient outcomes than standard chemotherapy. The characterization of tumors through analysis of prognostic and predictive biomarkers improves patient stratification and allows the identification of candidates for targeted therapies. Consequently, therapeutic decisions are ever more dependent on tumor molecular profiling and have a multidisciplinary approach, leading to a paradigm shift in the management of most cancers.

The current challenge in applying personalized oncology is the limited availability and quality of specimens for molecular analyses. Tumor tissue is still the gold standard for molecular analysis, and tissue biopsy has drawbacks such as invasiveness, partial representation of intratumoral and intermetastatic genetic heterogeneity, or unfeasibility for longitudinal monitoring. To overcome these limitations, multiple studies focused on discover new non-invasive biomarkers to characterize tumor-specific signatures based on multi-omics analysis that combines genomic, transcriptomic, and proteomic data [75].

Liquid biopsy has emerged as a minimally invasive diagnostic tool to analyze tumoral genetic biomarkers released into circulation [76]. It demands less time and costs for sample obtaining (mainly peripheral blood) than tissue biopsy, captures better the genetic heterogeneity, and gives a dynamic picture of the tumor molecular landscape. This concept englobes different analytes such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), RNA (mRNA and microRNA), and extracellular vesicles (EV). The main disadvantage is lack of standardization of methodologies, necessary for use in clinical practice. Recent publications in CCLM provide an update on this field, concerning issues that still need to be addressed and future perspectives and focusing particularly on frequent neoplasms, such as lung, breast and prostate cancer.

The implementation of liquid-biopsy biomarkers, particularly (ctDNA), has had a relevant impact on the management of patients with advanced non-small cell lung cancer (NSCLC), in which tumor genotyping is the standard of care. The analysis of activating mutations in EGFR gene is highly recommended because they are the most common druggable genetic alterations in lung adenocarcinomas and targeted treatment with EGFR-tyrosine kinase inhibitors (TKIs) has demonstrated improved survival. Unfortunately, it is not possible to obtain tumor tissue in up to 60% of patients with LC [77]. ctDNA measurement in blood and other fluids is an adequate source for EGFR testing [78], backed by oncology guidelines, to select treatment and also during the treatment monitoring to identify resistance mutations. The analysis of EGFR mutations in plasmatic ctDNA through cobas^® EGFR Mutation Test v2 was the first liquid-biopsy test approved by the FDA in 2016, but recently, multiple pan-cancer panels based on next-generation sequencing (NGS) obtained FDA approval. The analysis of ctDNA is also useful during the follow-up of patients treated with TKIs, for early identification of resistance mutations (T790M). The correlation between TMs and molecular features has been scarcely studied, and although recent studies show that the combination of conventional TMs and ctDNA could be a promising strategy, further studies are needed to elucidate the relationship between serum TM and gene mutations [79].

BC is another successful example of precision oncology application, as the better comprehension of BC molecular profiles has improved tumor characterization and also prognosis estimation. Nowadays, BC is classified into four molecular subtypes: Luminal A, Luminal B, HER2-enriched and Basal-like. In particular, HER2 overexpression occurs in approximately 20–30% of patients and is associated with more aggressive behavior and poor prognosis [80]. The development of anti-HER2 targeted treatments has improved overall survival of patients with this subtype of BC [81]. In this scenario, the HER-2 immunoassay in serum (sHER2), which measures the extracellular domain (ECD) of the protein, was approved in 2000 by the FDA. Although sHER2 showed low sensitivity compared with conventional TMs, their combination (CEA, CA 15.3 and HER2) increased the globall sensitivity and is especially useful when conventional TMs are negative [82]. This newest TM is scarcely mentioned in guidelines, only the NACB supports its potential role for monitoring the disease in patients with advanced HER2-positive BC. Preliminary results pointed out their value as a prognostic and therapeutic predictive factor [83, 84], but further studies are needed.

Moreover, patients with BC present increased levels of cfDNA in plasma, which has been associated with more aggressive or metastatic disease [73, 85]. Changes in the specific fraction of cfDNA derived from the tumor, the circulating tumor DNA (ctDNA), were also evaluated as a biomarker of response [86]. The detection of ctDNA mutations and serial quantification of the variant allele frequency (%VAF) during the follow-up could play a crucial role to monitor response and also to detect resistance mutations [76, 87].

In PCa, the quantification of microRNas (miRNas) in serum or plasma has shown promising results for diagnosis and risk stratification, as they can be useful for the inclusion of patients in active surveillance. Different panels of specific circulating miRNas have been evaluated in PCa, however, since there is no homogeneity in the miRNa used, the precision obtained for this biomarker is variable. Best results were obtained using miR-141, miR-375 and miR-21 but more studies are needed to verify its usefulness. Secondly, preanalytical conditions and miRNAs isolation play an important role in miRNAs measurement. Because of that, miRNAs were isolated from urine after prostate massage, opening a promising way for the management of early PCa [88].

Conclusions

This review provides an overview of the successes and pitfalls of TMs in the management of patients with cancer. We summarized the main obstacles to their use and remarked on the value of clinical guidelines to improve the quality of care received by patients, saving potential harm derived from the inappropriate use of these tests.

For safe and correct use, it is essential to know the limitations and diagnostic efficacy of each TM in different clinical situations. TM results should be evaluated considering confounding factors, concomitant clinical conditions, and potential causes of false positives. With this aim, laboratory physicians have a key role investigating discordant results, as well as facilitating correct interpretation.

Furthermore, we reported recent changes in the clinical usefulness of circulating TMs, and the new role of PSA in a personalized PCa screening strategy. We also reviewed recent studies that seek more specific and sensitive TMs focusing on those who have reached the clinical routine. HE4 is a clear example of a novel biomarker that has become a reality, improving risk stratification of patients with suspected ovarian cancer.

Finally, we have focused on potential applications of circulating biomarkers in precision oncology. We summarized current challenges in this field, and how non-invasive approaches can overcome some limitations. We discussed potential applications of liquid-biopsy biomarkers if real clinical integration is achieved. In this context, ctDNA have incorporated into clinical workflow, optimizing the management of cancer patients.