Tumor microenvironment and systemic disease: a dual target in medical oncology (also in the case of biomarkers)

In the current multidisciplinary management of cancer patients, the role of laboratory medicine is as indispensable as the role of surgical, medical or radiation oncology [1]. Much of the progress that we have witnessed during the last decades in the therapy of cancer patients is associated with the advent of targeted treatment. Apart from an anatomical targeting [2], the targeted therapy is critically dependent on the identification of a therapeutic target, i.e. a pathogenic mechanism or pathway distinguishing neoplastic from normal tissue that correspond to the hallmarks of cancer defined by Hanahan and Weinberg [3]. Biomarkers reflecting these hallmarks of cancer are of critical importance in guiding the targeted therapy.

In medical oncology, histological verification is an indispensable precondition before starting antitumor therapy. While the histological sample is usually obtained from the primary site, the real target of systemic therapy is not the primary tumor as much as the systemic disease (i.e. distant metastases) that usually represents the real threat for the patient’s life. Thus, the neoplastic disorder represents in reality a dual target, as we have to achieve the control of both the primary tumor and systemic disease that may be present as distant (micro)metastases.

In many instances, the targeted therapy is guided by empirical data. For example, although targeted therapy virtually transformed the management of metastatic renal cell carcinoma, the current treatment strategy consists of sequential administration of active drugs that is guided by accumulating clinical experience rather than tumor biomarkers [4]. In some tumors of other primary locations, the approach has been more target-oriented. With the advent of targeted therapy it has been recognized that common tumors such as non-small cell lung cancer (NSCLC) are in fact a group of different relatively less common molecularly-defined primary tumors as outlined in the review by Han and Li [5] in the present issue. One example illustrating the successful utilization of biomarkers in guiding the targeted therapy in NSCLC is the treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI). In patients with EGFR mutation the TKI therapy has shown improved efficacy and a more favorable toxicity profile compared to platinum-based cytotoxic therapy that is still currently the general standard of care in most NSCLC patients with tumors not harboring these mutations.

The analysis of the tumor tissue has traditionally been the backbone on which therapeutic decisions in the management of cancer patients have relied. Histological verification is still an indispensable part of diagnostic work-up before any anticancer therapy can be initiated. Predictive biomarkers like human epidermal growth factor receptor 2 (HER2) over-expression or BRAF mutations that are detected in tumor tissue guide the selection of therapeutic strategies in breast cancer or malignant melanoma. In breast cancer or melanoma usually “tissue is not the issue” as samples of sufficient size are relatively easily obtained from the primary and metastases are also often accessible for the biopsy. There are, however, situations when the necessity to obtain tumor tissue represents an important limitation for predictive diagnostics. This is particularly true for NSCLC as addressed in the review by Han and Li [5]. The field of NSCLC has advanced thanks to precision medicine, but the issue of a sample remains crucial here. Although tissue biopsy is the optimal sample, in many instances this is not possible and cytological biopsy or liquid biopsy may substitute for the lack of available tumor tissue.

In addition, two papers in the present issue report on the use of pleural effusion for the detection of EGFR mutations. It is not surprising that both of these studies come from East Asia where EGFR mutation rate is much higher than in the West. As the authors point out, the size of biopsy specimen may represent an important limitation for biomarker studies in patients with NSCLC. Pleural effusion is very common in patients with advanced NSCLC. Removal of pleural fluid is a therapeutic procedure and this material that would otherwise be discarded may provide an invaluable sample matrix for diagnostic purposes. Shin et al. [6] have studied pleural fluid samples in 77 NSCLC patients using the PCR method in supernatants and cell pellets. In total, EGFR mutation was detected in 35 (46%) of the patients. The detection rate was increased by PCR analysis of the supernatant, and the diagnostic yield was improved by 11%. The presence of EGFR mutation was detected even in samples with a very low quantity of DNA. The study highlights the importance of analyzing both supernatants and cell pellets. Importantly, although the data is rather anecdotal, the authors report the detection of resistance mutations. This could be of importance in patients progressing on EGFR TKI therapy.

Wang et al. [7] compared the detection of EGFR mutations using fluorophore-labeled peptide nucleic acid probes in pleural fluid with the detection in tumor tissue biopsies. The concordance rate was 80%. Similarly to the study reported by Shin et al. a comparison of the results using pleural fluid supernatants and cell pellets was made, and higher yield was observed in pellets. Again, the analysis of the sensitive or resistance mutations allowed for the prediction of response to targeted therapy. These two studies highlight the advantage of the analysis of tumor microenvironment in contrast to measuring biomarkers in the circulation. Finding biomarkers in the tumor microenvironment may allow an increase of the precision of targeted therapy although the target is also the systemic disease.

The method used to detect EGFR mutations could be essential as demonstrated in the paper by Martínez-Carretero et al. [8]. As the authors point out, a number of approaches are currently used in EGFR mutation testing that can be distinguished into screening methods that would detect any mutation and methods specific for known mutations. In this study, high resolution melting was used as the screening method and a commercial Cobas PCR test as a method to detect known mutations. Concordance between the two methods was found in 92% of the cases (56% of the positive results and 92% of the negative results). Massive parallel sequencing was used for the investigation for discordant results. Both high resolution melting and commercial Cobas PCR failed to detect mutations sensitive to anti-EGFR therapy in some patients. These data suggest that both methods should be used complementarily to avoid missing patients who might benefit from targeted therapy.

Obviously, the most common in clinical practice is the determination of circulating biomarkers as an aid in diagnosis or monitoring disease activity. Prominent among the circulating biomarkers has been the prostate specific antigen (PSA). In fact, the introduction of PSA has fundamentally transformed the management of prostate cancer. The communication by Diamandis et al. [9] introduces data that go beyond the traditional use of PSA. PSA is routinely assessed only in males. In females the concentrations were several orders of magnitude lower than in men and were not detectable with older-generation assays. With highly sensitive assays serum PSA concentrations may be measured also in women. Interestingly, as demonstrated by Diamandis et al. [9] serum PSA concentration markedly decreases with age. Future studies will determine what role, if any, could PSA play in the management of cancers in women, e.g. in breast cancer.

In recent years we have witnessed the emergence of immunotherapy as a fundamental approach in cancer treatment. For more than a century, immunotherapy of cancer has remained an elusive goal [10]. This is hardly surprising if we consider that systemic therapies, including immunotherapy, are administered in patients with advanced cancer who have a dysfunctional immune system [11], [12]. There is ample evidence of defective immune response in patients with metastatic cancer both systemically and in the tumor microenvironment [11], [12], [13], [14], [15]. On the other, some biomarkers of immune response in cancer patients like the tumor infiltration by lymphocytes predict not only prognosis, but also response to other therapeutic intervention, e.g. chemotherapy [16]. Biomarkers of the immune response may help in selecting patients for immunotherapy. If we take the example of NSCLC, similarly to the presence of EGFR mutations, the expression of programmed cell death receptor ligand (PD-L)-1 identifies a population of patients who benefit from the administration of pembrolizumab, an anti-PD-1 antibody compared to platinum-based chemotherapy [17].

The development of immunotherapy as an effective method of treatment in patients with cancer has been linked with the advent of monoclonal antibodies targeting immune checkpoints. Immune checkpoints are key regulators of the immune response, and the principal function of these molecules is to maintain the balance between elimination of invading organisms and avoiding autoimmunity. In cancer patients the blockade of immune checkpoints releases the immune response from the inhibition by the tumor that is now recognized as one of the hallmarks of cancer [3]. However, as other therapeutic methods, immune checkpoint blockade is not free of side effects, and these side effects are in fact caused by an autoimmune response to the host tissue. In general, autoimmunity has been traditionally a neglected topic in cancer medicine.

Two papers address the topic of autoimmunity in patients with cancer. Thyroid cancer is characterized by high cure rates by combined treatment including surgery and radioactive iodine therapy. Thyroglobulin is being used as a biomarker to detect tumor recurrence. In fact, the measurement of thyroglobulin is an essential component of disease management in patients with differentiated thyroid cancer. The presence of antibodies against thyroglobulin can cause false negative results of thyroglobulin immunoassays during thyroid cancer follow-up. This issue is addressed by a retrospective study of Katrangi et al. [18]. As the authors point out the cut-offs for assays for different thyroid antibodies have been defined for autoimmune diseases that are much more common than thyroid cancer, and the interpretation of the results in cancer patients may be difficult. When comparing four different thyroglobulin antibodies assays, considerable differences were observed adding further complexity to the issue. The combined determination of thyroglobulin and thyroglobulin antibody increased diagnostic sensitivity [18].

In addition to being of importance in monitoring patients after radioiodine therapy, anti-thyroglobulin antibodies may directly affect the biology of the disease. Trimboli et al. [19] present data from a large retrospective series indicating that the presence of anti-thyroglobulin antibodies before radioactive iodine therapy is associated with adverse outcome. This finding underscores that the immune response is in fact a double-edged sword in cancer patients as it could result in both the elimination of the tumor cell while under different conditions it could foster tumor growth. Immune-mediated thyroid disorders are prominent among the toxicity of monoclonal antibodies targeting immune checkpoints [17], [20], [21], and future studies should address the effect of these immunotherapeutic agents on anti-thyroglobulin antibodies and the association with outcome, including the toxicity.

What else can we learn from the papers highlighted by this editorial? In addition to the specific knowledge on EGFR mutations or autoimmune antibodies in thyroid cancer, these papers illustrate the need to study biomarkers both in the tumor microenvironment and systemically (i.e. in the circulation), including biomarkers of immune response, with a focus not only on biomarkers predicting the treatment outcome, but also biomarkers for monitoring and predicting treatment toxicity. The dualistic view of cancer as a local disease with the concept of tumor microenvironment and systemic metastases should also be reflected at the level of biomarker studies.

As indicated above, the study of biomarkers in the tumor microenvironment has been hampered by the need to obtain biopsy specimens, in the case of the investigation of biomarker dynamics in a repetitive fashion. From this perspective, malignant effusions like pleural effusion or ascites present with a unique opportunity for repetitive sampling. Unfortunately, malignant effusions as a sample matrix are often overlooked. In addition to the identification of biomarkers that characterize the tumor cell, the analysis of malignant effusions could be useful specifically in the monitoring of the host antitumor response [22]. For example, in malignant ascites of patients with advanced malignancies the phenotype of leukocytes consistent with defective antigen presentation and costimulatory pathway have been observed [12], [14]. Biomarkers of immune response, e.g. neopterin and kynurenine/tryptophan ratio, may also be studied in malignant effusions. Both neopterin synthesis and tryptophan degradation to kynurenine result from interferon-gamma induced metabolic pathways. Increased neopterin or kynurenine concentrations in this context reflect chronic stimulation of the immune response that is associated with immune dysfunction, both locally and systemically. While tryptophan depletion could inhibit tumor growth and kynurenine has, in high concentrations, direct antitumor activity [23], the predominant effect of tryptophan depletion and kynurenine synthesis is immune suppression.

The utilization of laboratory methods in the diagnosis and assessment of adverse events caused by anticancer therapy remains a neglected topic. While laboratory parameters are essential for the assessment of hematologic toxicity, the estimation of other side effects like gastrointestinal toxicity or skin toxicity still relies on symptoms reported by the patient or physical examination [24], [25], [26]. With the advent of novel immunotherapeutic agents, the need for biomarkers for monitoring or possibly predicting immune-related adverse events is becoming urgent. The toxicity of immunotherapy is predominantly systemic and these biomarkers should be of a systemic nature, including, e.g. antibodies like anti-thyroid antibodies.

In conclusion, the medical management of cancer is determined by aiming both at the primary tumor and systemic disease. In analogy, in biomarker detection that represents an increasingly complex topic we should not forget about this dual focus on the tumor microenvironment and systemic effects.