Sage Journals: Discover world-class research

Abstract

Owing to advances in treatment paradigms across the last five decades, testicular cancer is now eminently curable. However, current serum tumour and imaging biomarkers lack adequate sensitivity, specificity, and predictive value. Subsequently, their utility in detecting active malignancy and informing treatment decisions is minimal in a large proportion of men with testicular cancer. Micro-ribonucleic acids (miRNA), pertinently miR-371a-3p, offer a new tool, which based on early data, appears to fill many of the gaps that existing biomarkers leave. This paper reviews the evolution of the technology, potential limitations, and discusses the clinical relevance of miRNA as it moves towards the clinic.

Keywords

blood-based biomarker germ cell tumour molecular oncology predictive biomarker prognostic biomarker

Introduction

Testicular germ cell tumors (TGCT) are the most common malignancy diagnosed in young men in Western countries. Fortunately, these malignancies are considered highly curable.¹ However, treatment may result in significant, long-term morbidities in men with an otherwise excellent prognosis and there is room to improve the care of this population.

Almost two thirds of men diagnosed with TGCT will have disease confined to the testis at diagnosis (clinical stage 1, CS1), with the remainder having either regional nodal involvement (clinical stage 2, CS2) or distant spread (clinical stage 3, CS3). Following formal staging, subsequent treatment depends on histological subtype, disease stage, and prognostic group [as defined by the International Germ Cell Cancer Collaborative Group, (IGCCCG)], as well as patient factors and is guided by local and international guidelines.^1–4 While chemotherapy, radiotherapy, and surgery are undoubtedly very effective treatments, there is a growing panoply of evidence demonstrating the potential long-term morbidities associated with these therapies. In a study of survivors, high rates of obesity, sensory neuropathy, hypogonadism, erectile dysfunction, and cardiovascular disease^5,6 were reported. In addition, there is accumulating evidence showing a deleterious impact that these treatments have on health-related quality of life.^7–10 Surgical techniques are also not without their problems, with many men reporting problems with anejaculation and infertility; however, these may be lessened by modern, nerve-sparing techniques.^11–13 Therefore, it is integral that clinicians accurately select men who are most likely to benefit from treatment.

Despite widespread endorsement in surveillance guidelines,^1–4 current serum tumour and imaging biomarkers in TGCT offer limited sensitivity, specificity, and predictive value in detecting active disease,¹⁴ adding little value to treatment algorithms. Ultimately, there remains a number of important clinical questions that current tools fail to enlighten.

An ideal biomarker would help diagnose TGCT and accurately detect features of early relapse. It would reduce the issues associated with false positives seen with existing serum and imaging biomarkers, preventing unnecessary and potentially morbid treatments. An ideal biomarker would also detect minimal residual disease (MRD) post-orchidectomy and help select men who will most benefit from adjuvant treatment, rather than applying an all-or-nothing approach for this common clinical scenario. It would also help nuance the management of non-specific radiological changes and post-chemotherapy residual masses, differentiating fibrosis and necrosis from active germ cell tumors (GCT), including teratoma. In turn, it would eliminate pN0 retroperitoneal lymph node dissections (RPLND), sparing a proportion of men from this invasive procedure and directing this therapy to men with active GCTs who will derive benefit. An ideal biomarker would also help define treatment algorithms, including monitoring, and choice between treatment modalities in advanced disease, as well as inform prognosis and risk of relapse for men following treatment for TGCT. Clearly, there is room for improvement in a multitude of clinical scenarios.

There has been increasing interest in micro-ribonucleic acids (miRNA) as predictive biomarkers in various cancer types, with certain clusters of miRNAs present almost invariably in the blood of men with active TGCT^15–17 opening the door for further development. Early signs point towards miRNA being valuable biomarkers in the care of men with TGCT, filling some of the gaps that current biomarkers leave and offering accurate predictive information, improving the focus on the long-term health of this population. The evolution of knowledge regarding miRNA has been rapid with the majority of work undertaken by a few key research groups.

This paper will review the growing evidence base for miRNAs in the routine management of men with TGCT, discussing current limitations and applications in clinical practice.

Current serum tumour biomarkers and their deficiencies

Alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH) are variably expressed between histologic subtypes of TGCT and are the only existing serum biomarkers endorsed in the diagnosis and surveillance of men with this malignancy.¹⁸ The detection of these biomarkers in the serum of men with TGCT is explained by their embryological origins from gonocytes in normal development.¹⁹

Spermatogonia (and oogonia) arise from primordial germ cells contained within the extraembryonic mesoderm; these later become identifiable in the endoderm of the yolk sac.²⁰ AFP is produced by the embryological yolk sac in normal fetal development and by the liver later in gestation. It is, therefore, commonly measurable in the serum of men with yolk sac tumors (YST) and embryonal carcinoma (EC).²¹ In men with significant AFP elevations, YST must be suspected, even if it was not apparent in the original orchidectomy specimen.¹ Importantly, AFP may also be detectable in the serum of patients with hepatic disease, including hepatocellular carcinoma^21,22 and chronic liver disease secondary to alcohol misuse, viral hepatitis, and biliary tract disorders^18,21,as well as other gastrointestinal cancers,²³ inherited conditions such as hereditary ataxia-telangiectasia syndrome,²⁴ and, occasionally, specific drugs.¹⁸ It is rarely detected in seminoma or choriocarcinoma (CHC). The half-life of AFP is 5–7 days, which is important when interpreting elevated levels post-orchidectomy.²¹

The half-life of hCG is shorter and normally reduces within 12–36 h following orchidectomy. hCG is produced by syncytiotrophoblastic components in tumors, arising from trophoblasts associated with the placenta in embryological development. For this reason, elevation in hCG is commonly seen in CHC, and occasionally in pure seminoma, as well as other non-TGCT malignancies including neuroendocrine, bladder, renal, and lung carcinomas and non-malignant conditions including hypogonadism and tetrahydrocannabinol use.²¹

Of the existing serum tumor biomarkers, LDH is least specific for TGCT and is ubiquitously expressed by non-malignant cells, purely reflecting cell turnover. In malignancy, LDH is often measured at diagnosis as a surrogate for tumour bulk and risk of tumor lysis, and may be elevated in TGCTs, as well as non-malignant conditions including shock, liver disease, and muscle damage.^25–27

Ultimately, AFP, hCG, and LDH are only elevated in 26–34%, 38–47%, and 33–44% of men at diagnosis with any TGCT, respectively,²⁸ making their role in surveillance of men without elevation fraught. In seminoma, the value of serum tumor biomarkers is particularly low, with elevation of AFP, hCG, and LDH seen in <3%,^26,28–30 18–31%,^26,31 and ~30%^32,33 at diagnosis, respectively, and similarly low rates are also seen at the time of relapse are also seen.²¹ In fact, in the event of a significant elevation of serum tumor biomarkers in tumors otherwise thought to represent pure seminoma histologically, the diagnosis is revised from pure seminoma to NSGCT.^1,4 For this reason, serum tumor biomarkers are no longer strongly recommended as part of routine surveillance in some guidelines for men with seminoma,²¹ leaving clinicians relying on physical examination, modern imaging [including computerised tomography (CT) or magnetic resonance imaging, (MRI)] and symptoms as the only surveillance tools to detect relapse.

In NSGCT, serum tumor biomarkers are more commonly elevated. The degree of elevation often reflects clinical staging, with 10–20%, 20–40%, and 40–60% of men with NSGCT having elevation of AFP at diagnosis in CS1, CS2, and CS3, respectively. Elevation of hCG is slightly less common, seen in 10–20%, 20–30%, and 40%, respectively. LDH elevation is seen in 40–60% of patients across the disease spectrum.³² Despite this, <50% of men who relapse will have elevated serum tumour biomarkers,¹ with elevation more common if there was lymphovascular invasion in the original orchidectomy specimen.^34–36 Given the greater reliability in this population, however, serum tumor biomarkers continue to be used to determine IGCCCG prognostic risk classification and may influence treatment options.⁴ Despite this, these remain imperfect and subject to the issues outlined earlier.

Current imaging (bio)markers and their deficiencies

CT has long-formed part of the routine care in men with TGCT. It provides important structural information in the initial staging of malignancy and has a reasonable sensitivity in assessing advanced, untreated TGCT.³⁷ However, CT does have significant limitations. One of the major factors impacting the sensitivity of CT is its inability to detect malignancy in lymph nodes <1 cm, resulting in a missed opportunity to detect early relapse.⁴ Another key limitation is the failure of CT to differentiate between necrosis and fibrosis from active malignancy in post-chemotherapy residual masses in advanced disease. This is important, as men with persisting tumor masses at CT may go onto have post-chemotherapy RPLND (pcRPLND), only to find no viable malignancy or teratoma in the specimen (pN0), resulting in an unnecessary procedure.^38,39 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) may add additional value in this scenario.

In seminoma, FDG-PET offers improved sensitivity (80% versus 70%), specificity (100% versus 74%), and positive (PPV; 100% versus 37%) and negative predictive values (NPV; 96% versus 92%) for detecting viable malignancy in post-chemotherapy residual masses compared to CT and is routinely recommended in men with a residual mass >3 cm at least 6 weeks following chemotherapy.^40,41 There may also be a place for FDG-PET in the evaluation of men with seminoma experiencing rising serum tumour biomarkers following chemotherapy. In this area, FDG-PET offered a PPV of 92%, however the NPV is low, at just 50%.⁴² Unfortunately, FDG-PET has been shown to not be very useful in the evaluation of NSGCT,⁴³ nor in evaluation of small, non-specific lymph nodes across TGCT histologic subtypes.

The optimal frequency of CT surveillance is uncertain; however, it is important issue, given concerns regarding cumulative radiation exposure in this young population. A recent study⁴⁴ investigating an alternate surveillance schedule for CT in men with CS1 seminoma demonstrated that a reduced frequency of imaging was non-inferior to conventional surveillance. In their study, the risk of relapse beyond 36 months was <1%, providing reassurance that radiological surveillance may be safely reduced beyond this time point. In addition, MRI was non-inferior to CT, offering an attractive alternative approach and avoiding cumulative radiation exposure in well-resourced settings.⁴⁴

While imaging (bio)markers add useful clinical information for some men with TGCT, there remain a number of clinical scenarios where better biomarkers could revolutionize and personalize the care of these men. miRNAs are a promising biomarker in this space.

The evolution of microRNAs

miRNAs are small, non-coding ribonucleic acid molecules involved in the regulation of post-transcriptional gene expression. They contribute to important embryological functions, including organogenesis in normal development,^45–47 and have also been implicated in oncogenesis of various different solid organ and haematological malignancies. They interact closely with messenger RNA, affecting normal protein translation, and, when involved in oncogenesis, may act as either an oncogene or tumour suppressor gene.^48–51 Multiple adjacent miRNA genes considered ‘clusters,’ are recognised, and hold collective functions in normal development and oncogenesis; however, individual single miRNA molecules may, alone, hold the key to tumour growth, proliferation, and survival in some cancers.⁵²

In TGCT, miRNA-371 (miR-371) to -373 clusters are over-expressed by most histologic subtypes.¹⁷ Other miRNA types commonly seen in TGCT include miR-199a-3p, -302a-d, -214, -223-3, -367-3p, -383, -449, and -514a-3p. Adding weight to the integral role that miRNA may have in oncogenesis, specific miRNA recognised ubiquitously in TGCT are also present in tissue samples of germ cell neoplasia in situ, suggesting that over-expression may be an early step in oncogenesis.⁵³ Furthermore, miRNA clusters, for example miR-372-373, interfere with normal p53 function by interacting with the large tumor suppressor kinase 2 (LATS2) gene, inhibiting associated cyclin-dependent kinase (CDK) function, and leading to activation of the oncogenic Wnt/ß-catenin signaling pathway and uncontrolled cellular growth in TGCT development.^49,53–56

Other pathways to oncogenesis in TGCT include tumor suppressor miR-26a, and Let-7a, a miRNA precursor, which inhibit proliferation, migration, and invasive capacity in seminoma.⁵⁷ Importantly, these miRNA and miRNA precursors are downregulated in seminoma, leading to cell proliferation and growth.⁵⁸ miR-449 may also act as a tumour suppressor in TGCT, with its normal regulatory function embedded closely with CDK6 and the cell cycle. Concomitant retinoblastoma mutations in TGCT, however, lead to downregulation of miR-449, resulting in cell cycle progression and cell proliferation.⁵⁹ Furthermore, in EC, over-expression of miR-383 interferes with normal cell cycle regulation.^60–62 Given the increasingly clear role that miRNAs have in TGCT oncogenesis, these miRNAs and their oncogenic pathways will continue to attract attention and may become targets for drug development in the future.

However, there is some variability in tissue expression amongst TGCTs: teratoma demonstrates little-to-no expression of miR-371-373, miR-302, and miR-367, while seminomas are characterized by ‘average’ expression, and EC has extremely high expression. Importantly, these miRNAs are detectable in most histologic TGCT subtypes, regardless of primary site (gonadal versus extragonadal) and including ovarian primaries and across both pediatric and adult cases.¹⁷ While miRNAs are present in normal tissue, the expression profile of malignant tissue samples demonstrates relative dysregulation compared to their cell of origin, and importantly, the specific miRNAs that typify TGCT are not detectable in the serum in other cancers or disease states, which differs from our existing serum biomarkers.⁶³

Enveloped in an exosome, miRNAs resist breakdown by ribonucleases,⁶⁴ making them eminently measurable in serum, plasma, and other bodily fluids.^29,55,65,66 miRNAs are released into the bloodstream by malignant cells. This explains why higher concentrations of miRNA, specifically miR-371, are observed in testicular vein blood samples compared to the peripheral blood of men with TGCTs,⁵³ notwithstanding the fact that peripheral blood sampling is most practical in the clinical environment. Of the miRNAs detected in TGCT, miR-371a-3p, a member of the miR-371 cluster, has been most extensively studied, and appears to have the highest sensitivity and specificity of the biomarkers.^67,68 To-date, there has been no consensus as to the optimal way of measuring miRNA in peripheral blood, with practice varying significantly between the main research groups, including fundamentally which miRNA to routinely evaluate, use of serum or plasma analysis, assay choice, and cut-off values to define positivity.

When evaluating serum, peripheral blood is collected into serum separator tubes, and then centrifuged, aliquoted and frozen at −80°C while awaiting RNA extraction.⁶⁸ Instead, plasma is collected into cell-free deoxyribonucleic acid (DNA) tubes, containing an anticoagulant and cell preservative. Following collection, provided appropriate storage, samples may safely last several days, before being frozen as plasma aliquots and later undergoing RNA extraction. Plasma sampling offers additional advantages over serum, including the fact that circulating tumour DNA (ctDNA) may also be collected. This may be of increasing relevance as mutation profiles in TGCTs could have implications on diagnosis and treatment algorithms in the future.⁶⁹ In addition, isochromosome 12 (i12p), another evolving biomarker with high sensitivity and specificity for TGCT,^70–74 can be detected in ctDNA via in situ hybridization or next generation sequencing, which makes plasma an attractive medium. Importantly, hemolysis may vary between the analysis of miRNA in serum versus plasma, leading to unreliable results; as a consequence, this needs to be considered in sample processing and the interpretation of results.⁶⁸

The issue of cut-off values to define results is also an important consideration as miRNAs move towards the clinics. In the main studies, the relative quantity (RQ) of RNA defining positivity following quantitative polymerase chain reaction (PCR) has varied, with RQs of miR-371 between 2- and 5-times control, defined as positive. Dieckmann et al.⁷⁵ evaluated healthy blood donors and men with non-malignant testicular pathology and demonstrated equivalent, low RQs following PCR amplification in this population, whereas men with active TGCT had RQs of miR-371a-3p of >5. This cut-off was determined using a receive operator characteristic (ROC) analysis and Youden’s index. A consensus approach to measurement, analysis, and interpretation must be reached before this becomes a routine component of patient care.

Of the known miRNA, miR-371a-3p has been most comprehensively investigated in TGCT. Following orchidectomy for CS1 TGCT, serum miR-371a-3p reliably falls to <5% of pre-operative levels within 24 h,^55,76 with normalization within 6 days.^53,55,56 Its reported half-life is just <7 h.⁷⁶ As a diagnostic tool, miR-371a-3p also successfully discriminates active TGCTs from controls with a sensitivity of 91.8% and a specificity of 96.1%.⁷⁵ The sensitivity and specificity of miR-371a-3p does vary in different clinical scenarios; however, these high levels are generally seen across the board. As such, early data has determined the superiority of the assay when compared to conventional serum tumour biomarkers in detecting active TGCT.^75,77,78 From a health economics standpoint, early data supports the use of routine miRNA as a surveillance strategy.⁷⁹

Other studies have described the utility of miR-371 at diagnosis,^{14,53,56,76,80–82} in surveillance,^14,78,80,83 during treatment,^81,83,84 and in refining prognosis of TGCTs.⁸⁵ Unfortunately, many of these early studies lacked sufficient clinical follow-up to determine the full utility of these test in a real-world setting, providing impetus to embed this technology into randomised controlled trials to demonstrate its clinical utility.^86–88

Potential clinical application of miR-371

Given the shortfalls of our current biomarkers and early results with miR-371, there is clear appetite for miR-371 to enter routine practice. Pivotal work relating to the potential clinical utility of miR-371 has been conducted since 2011 and has been discussed extensively in the literature since. ^{57,67,68,71,89,90} The remainder of this paper will focus on the potential clinical applications of miRNA, specifically miR-371, and whether this tool is likely to become a routine component of care of men with TGCT.

Diagnosing TGCT

The most common presentation of TGCT is the development of a clinically apparent testicular mass. Thereafter, men go onto have further investigation, including testicular ultrasound, serum tumour biomarker evaluation, and CT to exclude metastatic disease.¹ Ultimately, the diagnosis is clinched on histopathological analysis of the orchidectomy specimen confirming TGCT.

As a diagnostic tool, conventional serum biomarkers are complementary to other tests, but lack adequate sensitivity and specificity.^25–27 In a variety of studies, miR-371 has been shown to have superior diagnostic value when compared to these biomarkers.^14,75,77,91 In a large study by Dieckmann et al.⁷⁵ serum miR-371a-3p offered a sensitivity, specificity, PPV, and NPV of 92%, 96%, 97%m and 83%, respectively, regardless of histologic subtype. Similarly high sensitivity and specificity was seen in seminoma (n = 323, 90% and 96% respectively) and NSGCT (n = 199, 95% and 96% respectively) making miR-371a-3p an attractive additional tool for the initial evaluation of men with TGCTs and offering higher diagnostic precision than older biomarkers. In addition, Dieckmann et al.⁷⁵ demonstrated a correlation between tumour size (pT1 versus pT2-4, p < 0.001), disease extent, (CS1 versus CS2-3 p < 0.001) and miR-371a-3p, such that the contemporary biomarker may also help accurately stage TGCTs and inform treatment recommendations. Given that up to 35% of men with CS2 TGCT may be down-staged to pathologic stage 1 at RPLND,^38,39,92 accurate staging is key, as it may significantly alter treatment recommendations, i.e., definitive chemotherapy, radiotherapy, or surgery versus active surveillance if deemed CS1.

A minority of patients diagnosed with GCTs will have an extragonadal primary, typically within the retroperitoneum or mediastinum, and rarely, primary intracranial GCTs. Despite anatomical variation, these tumours share similar embryologic origins with TGCTs,⁹³ and it is therefore reasonable to assume similar miRNA expression patterns to TGCTs. The accurate diagnosis of GCT is crucially important when evaluating men with extragonadal primaries, as, unlike most other non-GCT malignant histopathologies involving the retroperitoneal nodes, extragonadal GCTs may be offered curative-intent therapy. miRNAs may help to clarify this scenario when there is diagnostic uncertainty and avoid invasive biopsies.^29,94 In a small study, which evaluated serum miR-371, -372, -373, and -367 clusters in extracranial pediatric GCTs, including extragonadal and ovarian primaries, serum levels of these miRNA were significantly higher in patients with malignant GCTs when compared to patients with benign teratoma, other malignancies, and no known malignancy.²⁹ In particular, miR-371 offered an area under the curve (AUC) for diagnosis in a ROC analysis of 0.97 (p = 0.002), with other miRNA also offering a high level of diagnostic accuracy.

Intracranial GCTs pose a particularly unique clinical dilemma, given the significant risks associated with surgical biopsies required to refine the diagnosis. A small case series evaluating cerebrospinal fluid miR-371a-3p levels as a diagnostic tool for this group of patients showed that this tool may accurately diagnose GCT, sparing these patients from invasive biopsy.⁹⁵ These observations need to be further evaluated in a larger group of patients, ideally in a prospective manner, to inform clinical practice.

Recommending adjuvant treatment

One of the ongoing controversies in the care of men with CS1 TGCT is the role for adjuvant therapy, with a move away from routinely offering this treatment to all men with CS1 TGCT as was done historically. Following orchidectomy alone, most men with will be cured; however, a small proportion ultimately relapse and go onto require further treatment.¹ Adjuvant chemotherapy or radiotherapy may reduce the risk of relapse from ~20% to ~4% in seminoma, or ~30% to ~3% in NSGCT;^32,96 however, this comes with the perils of over-treating 70–80% of men who were destined to never relapse. While pathological features from the orchidectomy specimen may offer some insights into the risk of relapse,^{34–36,97–106} existing serum tumour biomarkers cannot identify men who will most benefit from adjuvant treatment. There has been a wide array of research into the role of miR-371 in detecting MRD following orchidectomy.^{14,56,75,107,108}

The largest study in this area was undertaken by Dieckmann et al.⁷⁵ They conducted a prospective, multicentric study including 256 men with CS1 seminoma and 112 men with CS1 NSGCT (74 mixed GCT, 29 EC, 3 YST, 6 teratoma), to evaluate the sensitivity and specificity of serum miR-371a-3p in this space. They observed a marked fall in measurable serum miR-371a-3p following orchidectomy (p < 0.001) in men with CS1 TGCT in 91.8% of patients. Unfortunately, this study lacked sufficient clinical follow-up to report upon the outcomes of the remaining ~8% of men who did not experience biochemical resolution of miR-371a-3p following orchidectomy. However, it is plausible that the ongoing elevation of the biomarker in these men may have represented MRD, making them more likely to relapse and also more likely to benefit from adjuvant therapy.

In contrast to these results, however, Lobo et al.¹⁰⁸ recently conducted a retrospective analysis of stored serum samples of 151 men with CS1 TGCT, including 101 with seminoma and 50 with NSGCT. In their cohort, which included relapses in 23% of men, post-orchidectomy serum miR-371a-3p levels or relative decline in serum levels post-orchidectomy did not accurately predict risk of relapse, suggesting that it may not be useful to guide decisions around adjuvant therapy. However, they did demonstrate the utility of miR-371a-3p in diagnosing relapse, with 94% of men experiencing elevation of the contemporary biomarker at relapse, compared to just 38% of men with elevation in AFP or hCG.

Clearly, miR-371 offers unique insights into the possible presence of active GCT, unlike existing biomarkers. However, prospective clinical trials are needed to further elucidate the potential role of the biomarker in refining recommendations for adjuvant therapy and detecting MRD.

Diagnosing relapse

While uncommon, the most frequent pattern of relapse in men originally diagnosed with CS1 TGCT is within retroperitoneal lymph nodes and for those with CS2+ disease at diagnosis, including extragonadal primary TGCTs, the first site of recurrence is commonly other nodal chains or visceral sites.¹ Reassuringly, the majority of men who do relapse can be offered curative-intent treatment with chemotherapy, radiotherapy, or surgery, depending on clinical characteristics and prior treatment. However, it is important to accurately diagnose relapse to ensure all available treatment options can be considered, and also, to reduce the risk of over-treatment and its associated toxicities.

In a multitude of studies, miR-371 has been shown to outperform existing biomarkers at the time of relapse.^{14,75,78,83,108} In one of the largest studies evaluating plasma miR-371a-3p in men with confirmed active TGCT,¹⁴ 44/46 evaluable patients had detectable plasma miR-371a-3p at relapse. The test offered a sensitivity of 96%, specificity of 100%, and NPV of 98%. It should be noted that the two patients who did not have detectable plasma miR-371a-3p had levels measured below the defined cut-off for ‘positivity’; the presence of active TGCT was later determined by histopathology (n = 1) or progressive conventional serum tumour biomarker elevation (n = 1), which speaks to the potential limitations of the new biomarker. Another study performed utilizing serum miR-371a-3p in this context also offered high sensitivity and specificity of 83% and 96%, respectively.⁷⁵ Furthermore, in a group of 28 men where the probability of recurrence was deemed to be ‘high-risk’ (90–100%) due to untreated, definitive regional, or distant TGCT, defined by conventional serum biomarkers or imaging, plasma miR-371a-3p diagnosed relapse with a sensitivity of 96%, specificity, and PPV of 100%; however, NPV in this analysis was lower at 66%.¹⁴

Serum and plasma miR-371a-3p appears to have reasonably high sensitivity, specificity, and predictive value in detecting relapse; however, larger, prospective trials are needed. Niche areas where miRNAs may add value includes the management of non-specific imaging changes, the evaluation of post-chemotherapy residual masses, and the identification of teratoma, where current tools leave significant gaps.

The management of non-specific radiological changes

Not infrequently, men will present with a surveillance-detected nodal mass with negative serum tumour biomarkers, leaving some doubt about the presence of active TGCT and resulting management approach. Depending on the extent of disease at the time of relapse, men may be committed to intensified surveillance, or alternatively one or more of chemotherapy, radiotherapy, or surgery.

Nappi et al.¹⁴ conducted an analysis of men with a prior diagnosis of CS1 TGCT, who had suspicious imaging findings, defined as 10–30 mm of nodal enlargement ± minor serum tumour biomarker elevation. They estimated that these men had a ‘moderate risk’ (25–50%) of harbouring active TGCT based on their clinical characteristics. Evaluating 34 men, they showed that plasma miR-371a-3p offered a sensitivity of 91%, specificity and PPV of 100%, and NPV of 96%. There was no apparent difference in the precision of plasma miR-371a-3p between seminoma and NSGCT in this analysis. Importantly, plasma miR-371a-3p also outperformed existing tools in these men, and those with CS1b NSGCT or post-chemotherapy residual masses with minor AFP elevation.¹⁴ In a ROC analysis for this whole population, the AUC for plasma miR-371a-3p was 0.89 [95% confidence interval (CI) 0.76–1.02] compared to 0.66 (95% CI 0.50–0.82) for CT, 0.65 (95% CI 0.48–0.84) for AFP, 0.61 (95% CI 0.43–0.81) for hCG, and 0.70 (95% CI 0.52–0.90) for LDH. While this data is compelling, the analysis may have been impacted by a short period of follow-up; hence, prospective evaluation with longer follow-up is required.

If miR-371 can accurately identify men with relapsed disease, particularly small-volume retroperitoneal nodal recurrences where existing tools leave considerable uncertainty, this will have a significant impact on the shape of care. While combination chemotherapy is an effective treatment for these men, there has been a move towards offering RPLND to men with small-volume retroperitoneal nodal recurrences of NSGCT⁴ and increasingly, seminoma,^109,110 due to the risk of long-term morbidity associated with systemic treatment.^5,6 Therefore, early diagnosis is key to avail all possible treatment options to men who do relapse.

The management of post-chemotherapy residual mass

Chemotherapy often results in substantial radiological reduction in tumour bulk; however, not uncommonly, a residual mass remains. With current tools, there continues to be uncertainty regarding the presence of active TGCT in residual masses. In seminoma, FDG-PET may help differentiate between residual tumour and fibrosis or necrosis in men with residual masses >3 cm.^40,41 In NSGCT, where FDG-PET is not helpful, these men will routinely be subjected to RPLND if they have persistent masses >1 cm, despite normalization of serum tumour biomarkers. In men undergoing this procedure, 40–45% will have residual teratoma and 10–15% will have viable, non-teratoma NSGCT. Despite this, the procedure still subjects up to 50% of men to major surgery from which they will derive no benefit.^111–113

In the largest study assessing the role of serum miRNA in detecting active NSGCT in post-chemotherapy residual masses, 82 men undergoing pcRPLND for this indication in a single, tertiary institution were evaluated.⁸¹ While conventional tumour biomarkers, specifically AFP and hCG, correlated well with disease stage and treatment response following chemotherapy, they were inadequate at detecting active disease in men who had a residual mass. In a group of 39 men with serial, serum miRNA samples pre- and post-chemotherapy, and following pcRPLND, miR-371a-3p, miR-373-3p, and miR-367-3p correlated well with residual active TGCT. Serum miR-371a-3p and -367-3p levels reliably fell during chemotherapy. In men whose pcRPLND specimen ultimately demonstrated fibrosis, necrosis, or teratoma only, no further reduction in miRNA levels was seen following surgery; however, men with active TGCT contained within the residual mass had a significant fall in measurable miRNA following pcRPLND. Notably, men with active TGCT in their pcRPLND specimen also had higher levels of miR-371, -373, and -367 post-chemotherapy than men who went onto have fibrosis, necrosis, or teratoma only. miR-371 had the highest discriminatory capability of the miRNA evaluated, with an AUC of the ROC 0.87 (95% CI 0.77–0.97, p < 0.0001). In a subgroup analysis of men with a residual mass up to 3 cm in largest axial diameter and without extra-retroperitoneal disease, miR-371a-3p accurately predicted men with active TGCT in their pcRPLND specimen with 100% sensitivity, 54% specificity, and 100% NPV (p = 0.02).⁸¹

These findings were corroborated in a smaller group by Nappi et al,¹⁴ who demonstrated a 100% sensitivity, specificity, and NPV in men with post-chemotherapy residual masses ± serum tumor biomarker elevation.

With such high sensitivity and NPV for detection of active NSGCT in post-chemotherapy residual masses, miR-371 clearly has tremendous potential to transform treatment paradigms for these men in the future. Less is known about the role for miR-371 in seminoma in this specific clinical context.

Using miRNA to evaluate teratoma

Given the risk of malignant transformation of benign teratoma, a classically chemotherapy- and radiotherapy-resistant pathology associated with a poor prognosis,¹ it is important to distinguish between teratoma, other active TGCT, and post-treatment fibrosis and necrosis. While miR-371 may help distinguish between fibrosis/necrosis and active non-teratoma NSGCT, it cannot adequately distinguish between fibrosis/necrosis and teratoma.⁸¹ Existing biomarkers are also unhelpful.⁹⁸ Given that miR-371 is not highly expressed by teratoma in serum or tissue, it is perhaps not surprising that miR-371 lacks sufficient sensitivity in the evaluation of this histologic subtype.¹⁷ Alternatively, there is preliminary evidence that an alternative miRNA cluster, miR-375, is expressed by teratoma,¹⁹ which may guide treatment decisions if clinically validated.

Nappi et al.¹¹⁴ conducted an exploratory study evaluating plasma miR-375 alone or integrated with miR-371a-3p in men with teratoma. In their initial analysis, plasma miR-375 was significantly higher in men with active teratoma, when compared to either CS1 GCT post orchidectomy during active surveillance (p = 0.01), or advanced seminoma (p = 0.04); and plasma miR-371a-3p was undetectable in both active teratoma and CS1 GCT post-orchidectomy during active surveillance (p < 0.0001). The resulting sensitivity, specificity, PPV, and NPV of plasma miR-375 for identifying teratoma were 0.90 (95% CI 0.69–0.97), 0.81 (95% CI 0.66–0.90), 0.69 (95% CI 0.50–0.83), and 0.94 (95% CI 0.81–0.98), respectively. However, in a validation cohort, miR-375 performed less well. When integrated with miR-371a-3p, the AUC of the ROC for miR-375 was 0.95 (95% CI 0.90–0.99), which was higher than either plasma miRNA alone.

In contrast, Lafin et al.¹¹⁵ were unable to demonstrate that miR-375, specifically miR-375-3p and -5p, was an effective biomarker in this space. They prospectively evaluated 40 pre-operative/post-chemotherapy serum samples of men undergoing pcRPLND for residual masses >1 cm. Histopathological review of pcRPLND samples was undertaken, confirming 19 teratomas, two mixed GCTs comprising teratoma, and either YST or EC, and 21 cases of fibrosis, necrosis, or benign lymph nodes. In their analysis, pre-operative serum miR-375-3p did not accurately predict the presence of teratoma in pcRPLND specimen, offering 86% sensitivity, 32% specificity, 58% PPV, and 67% NPV. Serum miR-375-5p at the same time point also lacked sensitivity (55%) and specificity (67%) in 20 men evaluated. Similarly, two other groups also found miR-375 had insufficient diagnostic value for teratoma,^116,117 with poor performance of miR-375-3p in a ROC analysis.¹¹⁶

Given the clinical relevance of this question, further prospective trials evaluating the role of miRNA clusters will be integral to answer this question for patients and the clinicians who care for them.

Treatment monitoring during chemotherapy

In men with serum tumour biomarker elevation at the time of relapse, the time to AFP and hCG normalization during chemotherapy has been shown to have prognostic value.^118–120 In a study which evaluated 653 men with advanced NSGCT from a collection of eight prospective trials, hCG normalization by week 3 favored an improvement in 4-year progression-free (PFS, p < 0.001) and overall survival (OS, p < 0.001). Normalization of AFP by week 3 similarly yielded an improvement in OS (p = 0.039), but not 4-year PFS (p = 0.054).¹¹⁹ As a result, there are ongoing studies relating to treatment intensification for men whose serum tumour biomarkers do not normalize by this time point.

In a similar fashion, a variety of studies have evaluated the natural history of miR-371 during chemotherapy, demonstrating a significant reduction in miR-371 levels during treatment, particularly after the first cycle of chemotherapy.^75,81,83 In an analysis of 70 men undergoing chemotherapy for CS2a and CS2b disease,⁷⁵ a significant fall in serum miR-371a-3p was seen following cycle 1, with a relatively smaller decline following cycle 2 and then plateau at normal levels. Clearly, serum miR-371a-3p is sufficiently sensitive to demonstrate treatment benefit; however, there is no correlative data which offers prognostic information in the same way that existing serum tumour biomarkers may in NSGCT. This too, is being evaluated in prospective trials.⁸⁸

Importantly, miRNA may also aid clinical decision-making where there is persistence of conventional serum tumour biomarkers and concern about chemo-resistance during treatment. In a single case report that reported a persistent rise in AFP during chemotherapy, serum miR-371a-3p and miR-367-3p were undetectable, and elevation of the conventional serum tumour biomarker was later explained by concomitant hepatic injury,¹²¹ providing clinicians further reassurance regarding the specificity of the contemporary biomarker in this context.

Choosing treatment modality in relapsed or advanced disease

Over the last five decades, there have been significant advances in the chemotherapy regimens, as well as surgical and radiotherapy techniques for men with advanced TGCT. In general, where there are multiple sites of disease identified on CT or solitary serum tumour biomarker elevation in NSGCT (usually S1), combination chemotherapy is the treatment of choice. However, where disease occurs in a single radiotherapy or surgical field, or serum tumour biomarkers are non-contributory, there remains some uncertainty around the ‘best’ treatment option, which may include any of the three modalities.⁸³ Presently, there is insufficient clinical data relating to the role that miRNA may have in this space; however, some studies have shed light on this issue.

Given that miR-371a-3p elevation has been correlated with tumour size and clinical stage,⁷⁵ it is possible that the biomarker may be utilized as a surrogate for disease extent. In the future, men with elevated miR-371a-3p without recurrence defined by existing serum tumour and imaging biomarkers may be recommended chemotherapy in the same way that men with CS1S NSGCT are currently treated. By the same rationale, chemotherapy may be preferred in men with disease traditionally defined as resectable in the event of significant miRNA elevation. These hypotheses need to be evaluated in prospective trials.

Plaza et al.⁸⁵ has shown that higher pre-chemotherapy serum miR-371a-3p, miR-373-3p, and miR-367-3p levels in men with advanced TGCT predicts a higher risk of relapse compared to men who had lower levels before chemotherapy, despite normalization of these miRNA during treatment and complete radiological response. It is plausible that men with significant miRNA elevation may benefit from treatment intensification with consolidative treatment or alternative chemotherapy regimens, however the cut-off value to define ‘high’ levels needs to be refined. In contrast, those with lower levels may benefit from de-intensification of treatment. This observation was replicated in another study, whereby men with ‘positive’ pre-chemotherapy plasma miR-371a-3p in the setting of advanced TGCT experienced an inferior PFS and OS than men with ‘negative’ miR-371a-3p and correlated these results with IGCCCG risk groups.⁸⁰ Clearly, there is room for improvement in this space.

Text box 1.

Current Serum Tumour and Imaging Biomarkers

The detection of serum tumour biomarkers reflects the embryologic origins of TGCT

Serum AFP, hCG, and LDH have poor sensitivity, specificity, and predictive value to diagnose TGCT, particularly seminoma

Serum tumour biomarker evaluation is no longer routinely recommended as part of surveillance in some guidelines for men with seminoma

Modern imaging, including CT and MRI, are unable to detect active malignancy in small lymph nodes; nor are they able to accurately differentiate between necrosis, fibrosis, and active malignancy

There is an established role for FDG-PET in evaluating post-chemotherapy residual masses >3 cm in seminoma

Text box 2.

MicroRNA

Small, non-coding ribonucleic acid molecules involved in post-transcriptional gene expression, interacting with messenger RNA, and may act as either an oncogene or tumour suppressor gene

Certain clusters of miRNAs typify TGCT, with miR-371-372 over-expressed by most histologic subtypes; others include miR-302a-d and miR-367-3p

Measurable in serum, plasma, and other bodily fluids; resist breakdown by ribonucleases

Significant variation in practice around sample collection methods, including the use of serum or plasma; with focus required to refine definitions of ‘positive’ and ‘negative’ miRNA results

Conclusions

miRNAs, particularly miR-371a-3p, are promising new biomarkers in the care of men with TGCT. The evolution of science underpinning miRNA in this space has been rapid, with significant advances in the technology and knowledge base across the last 10 years. In multiple studies, miR-371a-3p has been shown to be superior to existing serum tumour and imaging biomarkers. It has robust characteristics, with an apparent role in diagnosis and surveillance of TGCT, as well as offering prognostic information following orchidectomy and chemotherapy. Early evidence suggests that miR-371a-3p may fill many of the important gaps left by current diagnostic and surveillance tools. In teratoma, miR-375 may offer similarly helpful information; however, results have been conflicting to-date.

In addition to refining the above clinical scenarios, there is space to embed miRNA evaluation into routine surveillance, reducing the frequency of CT, as well as a possible place in treatment given the integral role miRNA play in the oncogenesis of TGCTs.^{49,53–62,122–125} Prospective trials evaluating the role of miRNA in CS1 TGCT, such as SWOG1823 and AGCT1531, are currently ongoing, however additional trials across the TGCT disease spectrum are required to validate existing datasets and further elucidate the role of this technology in the clinics.^86–88 If early data are able to be replicated in these prospective trials, serum miR-371a-3p has the ability to alter routine surveillance for many men, offering an inexpensive, safe, and precise tool that will personalize care and prevent over-treatment in a group of men with an otherwise excellent prognosis.

Text box 3.

Potential clinical applications of miRNA

Diagnosis: Serum miR-371a-3p offers sensitivity, specificity, PPV, and NPV of 91.8%, 96.1%, 97.2%, and 82.7%, respectively, in diagnosing TGCT regardless of histologic subtype and is superior to existing serum tumour biomarkers

Relapse: miR-371a-3p accurately detected relapsed disease in men with a confirmed relapse, with a sensitivity, specificity, and NPV of 96%, 100%, and 98% respectively

Adjuvant treatment: The role of miRNA in guiding use of adjuvant therapy is less clear. Some datasets have demonstrated that a minority of men with CS1 TGCT will not experience biochemical resolution of miR-371a-3p following orchidectomy, suggesting that they may harbor residual microscopic disease; however, Lobo et al. since demonstrated that post-orchidectomy miR-371a-3p levels did not accurately predict risk of relapse

Management of non-specific radiological changes: There appears to be a possible role for miRNA to finesse the management of non-specific radiological changes, with plasma miR-371a-3p accurately diagnosing active malignancy in men deemed at ‘moderate risk’ of harbouring TGCT with sensitivity, specificity, PPV, and NPV >90%

Post-chemotherapy residual masses: In men with post-chemotherapy residual masses >3 cm, miR-371a-3p accurately detected active TGCT with sensitivity and NPV of 100%; however, specificity was lower at just 54%

Teratoma: miR-371 seems less useful in the evaluation of teratoma, however a composite of miR-371a-3p and miR-375 may be helpful in diagnosing this condition

Treatment decisions and prognosis: miR-371a-3p falls reliably during chemotherapy, particularly after C1, however there is no correlative data relating the relationship between this observation and survival outcomes. There is also limited data pertaining to the way in which miRNA may guide treatment decisions, particularly around treatment intensification or consolidation and de-intensification. Early data has shown that higher levels of specific miRNA prior to chemotherapy may confer a worse prognosis than those with lower, albeit detectable levels at the same timepoint.

Footnotes

Conflict of interest statement

BT reports grants and personal fees from Amgen, grants and personal fees from Astra Zeneca, grants from Astellas, grants and personal fees from BMS, grants and personal fees from Janssen, grants and personal fees from Pfizer, grants and personal fees from MSD, grants and personal fees from Ipsen, personal fees from IQVIA, personal fees from Sanofi, personal fees from Tolmar, personal fees from Novartis, grants and personal fees from Bayer, and personal fees from Roche, outside the submitted work.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

References

National Comprehensive Cancer Network. Testicular Cancer (Version 2.2021). https://www.nccn.org/professionals/physician_gls/pdf/testicular.pdf (accessed 16 February 2021).

ANZUP. ANZUP surveillance recommendations for metastatic testicular cancer post-chemotherapy. https://anzup.org.au/docview.aspx?id=880 (2018, accessed 16 February 2021).

ANZUP. Stage 1 testicular cancer surveillance, https://www.anzup.org.au/docview.aspx?id=569 (2016, accessed 16 February 2021).

Stephenson

Eggener

Bass

, et al. Diagnosis and treatment of early stage testicular cancer: AUA guideline. J Urol 2019; 202: 272–281.

Haugnes

Wethal

Aass

, et al. Cardiovascular risk factors and morbidity in long-term survivors of testicular cancer: a 20-year follow-up study. J Clin Oncol 2010; 28: 4649–4657.

Kerns

Fung

Monahan

, et al. Cumulative burden of morbidity among testicular cancer survivors after standard cisplatin-based chemotherapy: a multi-institutional study. J Clin Oncol 2018; 36: 1505–1512.

Rossen

Pedersen

Zachariae

, et al. Health-related quality of life in long-term survivors of testicular cancer. J Clin Oncol 2009; 27: 5993–5999.

Vidrine

Hoekstra-Weebers

JEHM

Hoekstra

, et al. The effects of testicular cancer treatment on health-related quality of life. Urology 2010; 75: 636–641.

Chovanec

Vasilkova

Petrikova

, et al. Quality of life issues in long-term testicular germ cell tumor survivors. J Clin Oncol 2018; 36: e22107.

10.

Rudberg

Nilsson

Wikblad

Health-related quality of life in survivors of testicular cancer 3 to 13 years after treatment. J Psychosoc Oncol 2000; 18: 19–31.

11.

Stephenson

Tal

Sheinfeld

Adjunctive nephrectomy at post-chemotherapy retroperitoneal lymph node dissection for nonseminomatous germ cell testicular cancer. J Urol 2006; 176: 1996–1999; discussion 1999.

12.

Arai

Kawakita

Okada

, et al. Sexuality and fertility in long-term survivors of testicular cancer. J Clin Oncol 1997; 15: 1444–1448.

13.

Matos

Skrbinc

Zakotnik

Fertility in patients treated for testicular cancer. J Cancer Surviv 2010; 4: 274–278.

14.

Nappi

Thi

Lum

, et al. Developing a highly specific biomarker for germ cell malignancies: plasma miR371 expression across the germ cell malignancy spectrum. J Clin Oncol 2019; 37: 3090–3098.

15.

Murray

Nicholson

Coleman

Biology of childhood germ cell tumours, focussing on the significance of microRNAs. Andrology 2015; 3: 129–139.

16.

Palmer

Murray

Saini

, et al. Malignant germ cell tumors display common microRNA profiles resulting in global changes in expression of messenger RNA targets. Cancer Res 2010; 70: 2911–2923.

17.

Gillis

AJM

Stoop

Hersmus

, et al. High-throughput microRNAome analysis in human germ cell tumours. J Pathol 2007; 213: 319–328.

18.

Murray

Nicholson

JC.

α-Fetoprotein. Arch Dis Child Educ Pract Ed 2011; 96: 141–147.

19.

Shen

Shih

Hollern

, et al. Integrated molecular characterization of testicular germ cell tumors. Cell Rep 2018; 23: 3392–3406.

20.

Carlson

BM.

Extraembryonic membranes. In: Reference module in biomedical sciences. Elsevier, 2014.

21.

Gilligan

Seidenfeld

Basch

, et al. American Society of Clinical Oncology Clinical Practice Guideline on uses of serum tumor markers in adult males with germ cell tumors. J Clin Oncol 2010; 28: 3388–3404.

22.

Johnson

PJ.

Role of alpha-fetoprotein in the diagnosis and management of hepatocellular carcinoma. J Gastroenterol Hepatol 1999; 14(Suppl.): S32–S36.

23.

Schneider

Calaminus

Göbel

Diagnostic value of alpha 1-fetoprotein and beta-human chorionic gonadotropin in infancy and childhood. Pediatr Hematol Oncol 2001; 18: 11–26.

24.

Germà

Llanos

Tabernero

, et al. False elevations of alpha-fetoprotein associated with liver dysfunction in germ cell tumors. Cancer 1993; 72: 2491–2494.

25.

Ehrlich

Beck

SDW

Foster

, et al. Serum tumor markers in testicular cancer. Urol Oncol 2013; 31: 17–23.

26.

Dieckmann

K-P

Richter-Simonsen

Kulejewski

, et al. Testicular germ-cell tumours: a descriptive analysis of clinical characteristics at first presentation. Urol Int 2018; 100: 409–419.

27.

Lobo

Costa

Vilela-Salgueiro

, et al. Testicular germ cell tumors: revisiting a series in light of the new WHO classification and AJCC staging systems, focusing on challenges for pathologists. Hum Pathol 2018; 82: 113–124.

28.

Nørgaard-Pedersen

Schultz

Arends

, et al. Tumour markers in testicular germ cell tumours. Five-year experience from the DATECA Study 1976–1980. Acta Radiol Oncol 1984; 23: 287–294.

29.

Murray

Bell

Raby

, et al. A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours. Br J Cancer 2016; 114: 151–162.

30.

Milose

Filson

Weizer

, et al. Role of biochemical markers in testicular cancer: diagnosis, staging, and surveillance. Open Access J Urol 2011; 4: 1–8.

31.

Seidel

Daugaard

Nestler

, et al. Human chorionic gonadotropin-positive seminoma patients: a registry compiled by the global germ cell tumor collaborative group (G3). Eur J Cancer 2020; 132: 127–135.

32.

Kollmannsberger

Tandstad

Bedard

, et al. Patterns of relapse in patients with clinical stage I testicular cancer managed with active surveillance. J Clin Oncol 2015; 33: 51–57.

33.

Seidel

Daugaard

Nestler

, et al. The prognostic significance of lactate dehydrogenase levels in seminoma patients with advanced disease: an analysis by the Global Germ Cell Tumor Collaborative Group (G3). World J Urol. Epub ahead of print 8 March 2021. DOI: 10.1007/s00345-021-03635-3.

34.

Tandstad

Ståhl

Håkansson

, et al. One course of adjuvant BEP in clinical stage I nonseminoma mature and expanded results from the SWENOTECA group. Ann Oncol 2014; 25: 2167–2172.

35.

Ernst

Brasher

Venner

, et al. Compliance and outcome of patients with stage 1 non-seminomatous germ cell tumors (NSGCT) managed with surveillance programs in seven Canadian centres. Can J Urol 2005; 12: 2575–2580.

36.

Lago-Hernandez

Feldman

O’Donnell

, et al. A refined risk stratification scheme for clinical stage 1 NSGCT based on evaluation of both embryonal predominance and lymphovascular invasion. Ann Oncol 2015; 26: 1396–1401.

37.

Hilton

Herr

Teitcher

, et al. CT detection of retroperitoneal lymph node metastases in patients with clinical stage I testicular nonseminomatous germ cell cancer: assessment of size and distribution criteria. Am J Roentgenol 1997; 169: 521–525.

38.

Ghandour

Ashbrook

Freifeld

, et al. Nationwide patterns of care for stage II nonseminomatous germ cell tumor of the testicle. Eur Urol Oncol 2020; 3: 198–206.

39.

Heidenreich

Haidl

Paffenholz

, et al. Surgical management of complex residual masses following systemic chemotherapy for metastatic testicular germ cell tumours. Ann Oncol 2017; 28: 362–367.

40.

Bachner

Loriot

Gross-Goupil

, et al. 2-¹⁸fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) for postchemotherapy seminoma residual lesions: a retrospective validation of the SEMPET trial. Ann Oncol 2012; 23: 59–64.

41.

De Santis

Becherer

Bokemeyer

, et al. 2-18fluoro-deoxy-D-glucose positron emission tomography is a reliable predictor for viable tumor in postchemotherapy seminoma: an update of the prospective multicentric SEMPET trial. J Clin Oncol 2004; 22: 1034–1039.

42.

Hain

O’Doherty

Timothy

, et al. Fluorodeoxyglucose positron emission tomography in the evaluation of germ cell tumours at relapse. Br J Cancer 2000; 83: 863–869.

43.

Huddart

O’Doherty

Padhani

, et al. 18fluorodeoxyglucose positron emission tomography in the prediction of relapse in patients with high-risk, clinical stage I nonseminomatous germ cell tumors: preliminary report of MRC Trial TE22—the NCRI Testis Tumour Clinical Study Group. J Clin Oncol 2007; 25: 3090–3095.

44.

Joffe

Cafferty

Murphy

, et al. Imaging modality and frequency in surveillance of stage I seminoma testicular cancer: results from a randomized, phase III, factorial trial (TRISST). J Clin Oncol 2021; 39(Suppl. 6). doi: 10.1200/JCO.2021.39.6_suppl.374.

45.

Ahmad

Non-coding RNAs: a tale of junk turning into treasure. Noncoding RNA Res 2016; 1: 1–2.

46.

Boland

CR.

Non-coding RNA: it’s not junk. Dig Dis Sci 2017; 62: 1107–1109.

47.

Gross

Kropp

Khatib

MicroRNA signaling in embryo development. Biology 2017; 6: 34.

48.

Reis

Pereira

Lopes-Cendes

, et al. MicroRNAs: a new paradigm on molecular urological oncology. Urology 2010; 76: 521–527.

49.

Voorhoeve

le Sage

Schrier

, et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006; 124: 1169–1181.

50.

Esquela-Kerscher

Slack

FJ.

Oncomirs – microRNAs with a role in cancer. Nat Rev Cancer 2006; 6: 259–269.

51.

Hwang

H-W

Mendell

JT.

MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 2006; 94: 776–780.

52.

Wang

Yang

G-H

, et al. Human microRNA clusters: genomic organization and expression profile in leukemia cell lines. Biochem Biophys Res Commun 2006; 349: 59–68.

53.

Dieckmann

K-P

Spiekermann

Balks

, et al. MicroRNAs miR-371-3 in serum as diagnostic tools in the management of testicular germ cell tumours. Br J Cancer 2012; 107: 1754–1760.

54.

Zhou

A-D

Diao

L-T

, et al. β-Catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway. Oncogene 2012; 31: 2968–2978.

55.

Spiekermann

Belge

Winter

, et al. MicroRNA miR-371a-3p in serum of patients with germ cell tumours: evaluations for establishing a serum biomarker. Andrology 2015; 3: 78–84.

56.

Belge

Dieckmann

K-P

Spiekermann

, et al. Serum levels of microRNAs miR-371-3: a novel class of serum biomarkers for testicular germ cell tumors? Eur Urol 2012; 61: 1068–1069.

57.

De Martino

Chieffi

Esposito

. miRNAs and biomarkers in testicular germ cell tumors: an update. Int J Mol Sci 2021; 22: 1380.

58.

De Martino

Esposito

Pellecchia

, et al. HMGA1-regulating microRNAs Let-7a and miR-26a are downregulated in human seminomas. Int J Mol Sci 2020; 21: 3014.

59.

Yong-Ming

Ai-Jun

Xiao-Yue

, et al. miR-449a: a potential therapeutic agent for cancer. Anticancer Drugs 2017; 28: 1067–1078.

60.

Alsamman

El-Masry

OS.

Interferon regulatory factor 1 inactivation in human cancer. Biosci Rep 2018; 38: BSR20171672.

61.

Tian

Cao

Y-X

Zhang

X-S

, et al. The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis. Cell Death Dis 2013; 4: e617.

62.

Huang

Tian

Duan

, et al. microRNA-383 impairs phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest in testicular embryonal carcinoma cells. Cell Signal 2014; 26: 903–911.

63.

Novotny

Belling

Bramsen

, et al. microRNA expression profiling of carcinoma in situ cells of the testis. Endocr Relat Cancer 2012; 19: 365–379.

64.

Chim

SSC

Shing

TKF

Hung

ECW

, et al. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem 2008; 54: 482–490.

65.

Dieckmann

K-P

Spiekermann

Balks

, et al. MicroRNA miR-371a-3p - a novel serum biomarker of testicular germ cell tumors: evidence for specificity from measurements in testicular vein blood and in neoplastic hydrocele fluid. Urol Int 2016; 97: 76–83.

66.

Radtke

Dieckmann

K-P

Grobelny

, et al. Expression of miRNA-371a-3p in seminal plasma and ejaculate is associated with sperm concentration. Andrology 2019; 7: 469–474.

67.

Almstrup

Lobo

Mørup

, et al. Application of miRNAs in the diagnosis and monitoring of testicular germ cell tumours. Nat Rev Urol 2020; 17: 201–213.

68.

Murray

Huddart

Coleman

The present and future of serum diagnostic tests for testicular germ cell tumours. Nat Rev Urol 2016; 13: 715–725.

69.

Litchfield

Summersgill

Yost

, et al. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours. Nat Commun 2015; 6: 5973.

70.

Lianidou

Pantel

Liquid biopsies. Genes Chromosomes Cancer 2019; 58: 219–232.

71.

Lobo

Leão

Jerónimo

, et al. Liquid biopsies in the clinical management of germ cell tumor patients: state-of-the-art and future directions. Int J Mol Sci 2021; 22: 2654.

72.

Chovanec

Kalavska

Mego

, et al. Liquid biopsy in germ cell tumors: biology and clinical management. Expert Rev Mol Diagn 2020; 20: 187–194.

73.

Freitag

Sukov

Bryce

, et al. Assessment of isochromosome 12p and 12p abnormalities in germ cell tumors using fluorescence in situ hybridization, single-nucleotide polymorphism arrays, and next-generation sequencing/mate-pair sequencing. Hum Pathol 2021; 112: 20–34.

74.

Fichtner

Richter

Filmar

, et al. The detection of isochromosome i(12p) in malignant germ cell tumours and tumours with somatic malignant transformation by the use of quantitative real-time polymerase chain reaction. Histopathology 2021; 78: 593–606.

75.

Dieckmann

K-P

Radtke

Geczi

, et al. Serum levels of microRNA-371a-3p (M371 test) as a new biomarker of testicular germ cell tumors: results of a prospective multicentric study. J Clin Oncol 2019; 37: 1412–1423.

76.

Radtke

Hennig

Ikogho

, et al. The novel biomarker of germ cell tumours, micro-RNA-371a-3p, has a very rapid decay in patients with clinical stage 1. Urol Int 2018; 100: 470–475.

77.

Dieckmann

K-P

Radtke

Spiekermann

, et al. Serum levels of microRNA miR-371a-3p: a sensitive and specific new biomarker for germ cell tumours. Eur Urol 2017; 71: 213–220.

78.

Van Agthoven

Eijkenboom

WMH

Looijenga

LHJ

. microRNA-371a-3p as informative biomarker for the follow-up of testicular germ cell cancer patients. Cell Oncol 2017; 40: 379–388.

79.

Charytonowicz

Aubrey

Bell

, et al. Cost analysis of noninvasive blood-based microRNA testing versus CT scans for follow-up in patients with testicular germ-cell tumors. Clin Genitourin Cancer 2019; 17: e733–e744.

80.

Mego

Van Agthoven

Gronesova

, et al. Clinical utility of plasma miR-371a-3p in germ cell tumors. J Cell Mol Med 2019; 23: 1128–1136.

81.

Leão

Van Agthoven

Figueiredo

, et al. Serum miRNA predicts viable disease after chemotherapy in patients with testicular nonseminoma germ cell tumor. J Urol 2018; 200: 126–135.

82.

Anheuser

Radtke

Wülfing

, et al. Serum levels of microRNA371a-3p: a highly sensitive tool for diagnosing and staging testicular germ cell tumours: a clinical case series. Urol Int 2017; 99: 98–103.

83.

Terbuch

Adiprasito

Stiegelbauer

, et al. MiR-371a-3p serum levels are increased in recurrence of testicular germ cell tumor patients. Int J Mol Sci 2018; 19: 3130.

84.

Dieckmann

K-P

Frey

Lock

Contemporary diagnostic work-up of testicular germ cell tumours. Nat Rev Urol 2013; 10: 703–712.

85.

Plaza

Van Agthoven

Meijer

, et al. miR-371a-3p, miR-373-3p and miR-367-3p as serum biomarkers in metastatic testicular germ cell cancers before, during and after chemotherapy. Cells 2019; 8: 1221.

86.

Clinicaltrials.gov. A study of miRNA 371 in patients with germ cell tumors. US National Library of Medicine, 2021. https://clinicaltrials.gov/ct2/show/NCT04435756

87.

Children’s Oncology Group. AGCT1531: a phase 3 study of active surveillance for low risk and a randomized trial of carboplatin vs. cisplatin for standard risk pediatric and adult patients with germ cell tumors, https://clinicaltrials.gov/ct2/show/NCT04435756 (2021, accessed 23 March 2021).

88.

Clinicaltrials.gov. Accelerated v’s standard BEP chemotherapy for patients with intermediate and poor-risk metastatic germ cell tumours (P3BEP), https://clinicaltrials.gov/ct2/show/NCT02582697 (2020, accessed 13 April 2021).

89.

Lafin

Murray

Coleman

, et al. The road ahead for circulating microRNAs in diagnosis and management of testicular germ cell tumors. Mol Diagn Ther 2021; 25: 269–271.

90.

Liu

Lian

, et al. The diagnostic accuracy of miR-371a-3p for testicular germ cell tumors: a systematic review and meta-analysis. Mol Diagn Ther 2021; 25: 273–281.

91.

Badia

Abe

Wong

, et al. Real-world application of pre-orchiectomy miR-371a-3p test in testicular germ cell tumor management. J Urol 2021; 205: 137–144.

92.

Yadav

Retroperitoneal lymph node dissection: an update in testicular malignancies. Clin Transl Oncol 2017; 19: 793–798.

93.

Ronchi

Cozzolino

Montella

, et al. Extragonadal germ cell tumors: not just a matter of location. A review about clinical, molecular and pathological features. Cancer Med 2019; 8: 6832–6840.

94.

Murray

Halsall

Hook

, et al. Identification of microRNAs from the miR-371~373 and miR-302 clusters as potential serum biomarkers of malignant germ cell tumors. Am J Clin Pathol 2011; 135: 119–125.

95.

Murray

Ajithkumar

Harris

, et al. Clinical utility of circulating miR-371a-3p for the management of patients with intracranial malignant germ cell tumors. Neurooncol Adv 2020; 2: vdaa048.

96.

Hanna

Einhorn

LH.

Testicular cancer – discoveries and updates. N Engl J Med 2014; 371: 2005–2016.

97.

Mortensen

Lauritsen

Gundgaard

, et al. A nationwide cohort study of stage I seminoma patients followed on a surveillance program. Eur Urol 2014; 66: 1172–1178.

98.

Aparicio

Maroto

Del Muro

, et al. Prognostic factors for relapse in stage I seminoma: a new nomogram derived from three consecutive, risk-adapted studies from the Spanish Germ Cell Cancer Group (SGCCG). Ann Oncol 2014; 25: 2173–2178.

99.

Chung

Warde

Stage I seminoma: adjuvant treatment is effective but is it necessary?

J Natl Cancer Inst 2011; 103: 194–196.

100.

Chung

Daugaard

Tyldesley

, et al. Evaluation of a prognostic model for risk of relapse in stage I seminoma surveillance. Cancer Med 2015; 4: 155–160.

101.

Tandstad

Ståhl

Dahl

, et al. Treatment of stage I seminoma, with one course of adjuvant carboplatin or surveillance, risk-adapted recommendations implementing patient autonomy: a report from the Swedish and Norwegian Testicular Cancer Group (SWENOTECA). Ann Oncol 2016; 27: 1299–1304.

102.

Warde

Specht

Horwich

, et al. Prognostic factors for relapse in stage I seminoma managed by surveillance: a pooled analysis. J Clin Oncol 2002; 20: 4448–4452.

103.

Leman

Gonzalgo

ML.

Prognostic features and markers for testicular cancer management. Indian J Urol 2010; 26: 76–81.

104.

Nazeer

Kee

, et al. Spermatic cord contamination in testicular cancer. Mod Pathol 1996; 9: 762–766.

105.

Kobayashi

Saito

Kitamura

, et al. Oncological outcomes in patients with stage I testicular seminoma and nonseminoma: pathological risk factors for relapse and feasibility of surveillance after orchiectomy. Diagn Pathol 2013; 8: 57.

106.

Sturgeon

Moore

Kakiashvili

, et al. Non-risk-adapted surveillance in clinical stage I nonseminomatous germ cell tumors: the Princess Margaret Hospital’s experience. Eur Urol 2011; 59: 556–562.

107.

Nappi

Thi

Saxena

, et al. Longer follow-up data of circulating miR371a-3p expression across the spectrum of germ cell tumors (GCT). J Clin Oncol 2021; 39(Suppl. 6): abstract 379.

108.

Lobo

Leão

Gillis

AJM

, et al. Utility of serum miR-371a-3p in predicting relapse on surveillance in patients with clinical stage I testicular germ cell cancer. Eur Urol Oncol 2021; 4: 483–491.

109.

Albers

Hiester

Siemer

, et al. The PRIMETEST trial: interim analysis of a phase II trial for primary retroperitoneal lymph node dissection (RPLND) in stage II A/B seminoma patients without adjuvant treatment. J Clin Oncol 2019; 37: 507.

110.

Daneshmand

Cary

Masterson

, et al. SEMS trial: result of a prospective, multi-institutional phase II clinical trial of surgery in early metastatic seminoma. J Clin Oncol 2021; 39(Suppl. 6): abstract 375.

111.

Steyerberg

Keizer

Fosså

, et al. Prediction of residual retroperitoneal mass histology after chemotherapy for metastatic nonseminomatous germ cell tumor: multivariate analysis of individual patient data from six study groups. J Clin Oncol 1995; 13: 1177–1187.

112.

Fosså

Aass

Ous

, et al. Histology of tumor residuals following chemotherapy in patients with advanced nonseminomatous testicular cancer. J Urol 1989; 142: 1239–1242.

113.

Carver

Serio

Bajorin

, et al. Improved clinical outcome in recent years for men with metastatic nonseminomatous germ cell tumors. J Clin Oncol 2007; 25: 5603–5608.

114.

Nappi

Thi

Adra

, et al. Integrated expression of circulating miR375 and miR371 to identify teratoma and active germ cell malignancy components in malignant germ cell tumors. Eur Urol 2021; 79: 16–19.

115.

Lafin

Kenigsberg

Meng

, et al. Serum small RNA sequencing and miR-375 assay do not identify the presence of pure teratoma at postchemotherapy retroperitoneal lymph node dissection. Eur Urol Open Sci 2021; 26: 83–87.

116.

Belge

Grobelny

Matthies

, et al. Serum level of microRNA-375-3p is not a reliable biomarker of teratoma. In Vivo (Athens, Greece) 2020; 34: 163–168.

117.

Lobo

Gillis

AJM

Van Den Berg

, et al. Identification and validation model for informative liquid biopsy-based microRNA biomarkers: insights from germ cell tumor in vitro, in vivo and patient-derived data. Cells 2019; 8: 1637.

118.

Mazumdar

Bajorin

Bacik

, et al. Predicting outcome to chemotherapy in patients with germ cell tumors: the value of the rate of decline of human chorionic gonadotrophin and alpha-fetoprotein during therapy. J Clin Oncol 2001; 19: 2534–2541.

119.

Fizazi

Culine

Kramar

, et al. Early predicted time to normalization of tumor markers predicts outcome in poor-prognosis nonseminomatous germ cell tumors. J Clin Oncol 2004; 22: 3868–3876.

120.

Fizazi

Flechon

le Teuff

, et al. Mature results of the GETUG 13 phase III trial in poor-prognosis germ-cell tumors (GCT). J Clin Oncol 2016; 34: 4504.

121.

Lembeck

Puchas

Hutterer

, et al. MicroRNAs as appropriate discriminators in non-specific alpha-fetoprotein (AFP) elevation in testicular germ cell tumor patients. Noncoding RNA 2020; 6: 2.

122.

Cheng

Byrom

Shelton

, et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 2005; 33: 1290–1297.

123.

Murray

Coleman

MicroRNA dysregulation in malignant germ cell tumors: more than a biomarker?

J Clin Oncol 2019; 37: 1432–1435.

124.

Liu

Lian

Zhang

, et al. MicroRNA-302a sensitizes testicular embryonal carcinoma cells to cisplatin-induced cell death. J Cell Physiol 2013; 228: 2294–2304.

125.

Liu

Shi

, et al. miR-223-3p regulates cell growth and apoptosis via FBXW7 suggesting an oncogenic role in human testicular germ cell tumors. Int J Oncol 2017; 50: 356–364.

Improving outcomes in germ cell cancers using miRNA

Abstract

Keywords

Introduction

Current serum tumour biomarkers and their deficiencies

Current imaging (bio)markers and their deficiencies

The evolution of microRNAs

Potential clinical application of miR-371

Diagnosing TGCT

Recommending adjuvant treatment

Diagnosing relapse

The management of non-specific radiological changes

The management of post-chemotherapy residual mass

Using miRNA to evaluate teratoma

Treatment monitoring during chemotherapy

Choosing treatment modality in relapsed or advanced disease

Conclusions

Footnotes

Conflict of interest statement

Funding

References