Recommended approach for biomarkers in testicular cancer

Prof. Maria Pilar Laguna Pes Istanbul Medipol University Department of Urology Istanbul (TR)

plaguna@ medipol.edu.tr

Testicular cancer (TC) is a relatively rare cancer compared to the incidence of other urological cancers in males. However, it is the most frequent solid cancer in male adolescents and young adults with an, as yet, unquantified social, professional, and family related impact. The good news is that around 70% of testis tumours are diagnosed at an early stage and that the overall mortality is low. Nevertheless, the price to pay for cure may be high when considering the long life expectancy of these young patients. Hence, early detection, monitoring of response to primary treatment and recurrence, are of major importance.

This article aims to elucidate the current policy of the 2022 EAU TC Guidelines regarding the use of biomarkers in the diagnostic work-up and monitoring of TC, in the current context of so many emerging molecular biomarkers. Key question is rather, ‘Why do the 2022 Guidelines not recommend the use of micro RNAs (miR) in the diagnostic, treatment and follow-up pathways of TC patients and are the old recommendations still in place? Even though EAU22 includes a session on TC entitled “Is it prime time for biomarker-triggered follow-up?”

Before embarking on a discussion about replacing a set of markers that have been used as a standard to guide diagnosis, management or follow-up of a rare disease for almost five decades, it may be helpful to refresh two concepts; ‘What are the key attributes of a quality Clinical Practice Guideline (CPG)’, and ‘Which criteria apply for a biomarker to be accepted as a new standard of care?’.

CPGs aim to provide recommendations based on the best available evidence; providing a strength rating reflecting the certainty and evidence level of a given recommendation. They must be based on a critical, transparent, and reproducible methodology although guidelines can include expert opinion and consensus finding for areas with very diverse evidence or where no evidence exists.

Given the variation in health care systems across Europe and world-wide, cost-effectiveness assessment is not addressed in EAU guidelines. Rather than providing an overview of all available tests, the Guidelines focus on the minimum level of care to be provided, solely based on the clinical effectiveness of various tests. Ultimate aim is to promote implementation and standardisation of care.

Secondly, a universally applicable and accepted biomarker should ideally be non-invasive, i.e., be detectable in biological fluids such as urine or blood, and be easy to measure. Biomarkers must be capable of discriminating between healthy individuals (without disease) and those with the condition, be sensitive enough for the purpose of early diagnosis or detecting progression, with a long-serum half-life for the purpose of late diagnosis; be cost-effective with efficacy shown in prospective clinical trials, including sufficient patients numbers.

Last but not least, biomarkers should be able to play a role in a screening setting and ultimately reduce mortality.

The current biomarkers Currently, the EAU TC Panel recommend serum biomarkers ∝-fetoprotein (AFP) and the beta subunit of human chorionic gonadotropin (β-hCG). AFP is a long glycoprotein of the group of the serum albumins, produced mostly by the yolk sac, with a serum half-life of 5-7 days. β-hCG is a peptide hormone with a serum half-life of 18-36 hours and is expressed by the syncytiotrophoblast cells of some testicular germ cell tumours (TGCT), mainly by choriocarcinoma and embryonal carcinoma but it is also elevated in some seminomas. A third marker, lactate dehydrogenase (LDH) is an unspecific marker of cell death with a half-life of around 24 hours. Although it has limited value in the primary diagnosis, LDH still plays a role in the risk-classification of the International Germ Cell Cancer Collaboration Group (IGCCCG) for metastatic disease, always in conjunction with the two TCspecific markers. These markers are used in the diagnosis, prognosis, detection of recurrence, and to monitor the response to systemic treatment.

Overall, elevation of one or two of these markers is seen in up to 60% of patients at diagnosis. The presence of the TC-specific markers increases with the clinical stage (CS) from 50.2% (44-56%) in CS I to 93% in CS III (75.8-98.8%). An increase of both AFP and β-hCG is detected in up to 39% of patients with non-seminomatous germ cell tumour (NSGCT), and around 90% of NSGCTs present with a rise in either AFP or β-hCG at diagnosis. Pure seminomas may also have modestly elevated β-hCG level at diagnosis in up to 30% (9-32%) of patients.

Following orchidectomy, it takes several weeks for the serum markers to normalise according to their half-life (≈ 3 weeks). As a rule, the persistence of elevation or the increase in serum tumour marker level after orchidectomy indicates the presence of metastatic disease, although a slight elevation above upper normal limits may also be caused by other conditions. Conversely, although normalisation of marker levels after orchidectomy is a favourable indicator, it does not exclude the possibility of metastatic disease. Overall sensitivity is around 50-60% and specificity, positive-predictive value (PPV) and negative-predictive value (NPV) are low in the primary diagnostic setting and lower in seminoma than in non-seminoma TGCTs.

“...the Guidelines focus on the minimum level of care to be provided, solely based on the clinical effectiveness of various tests. Ultimate aim is to promote implementation and standardisation of care.“

In cases of metastatic TC, risk stratification is based on serum tumour marker levels immediately before initiation of systemic treatment. Before chemotherapy AFP and LDH levels may act as prognostic factors for OS in the non-seminoma intermediate-risk group. Although these tumour markers are routinely used for follow-up, only 25% of patients present with elevated AFP and β-hCG at relapse, and LDH may remain persistently elevated in 30% of patients despite cure. Most of the relapses are routinely diagnosed by imaging during follow-up.

Both in primary diagnosis or during follow-up after orchiectomy, mildly elevated non-rising AFP (< 20 ng/ ml) or β-hCG (< 20 IU/L) may not indicate presence of TGCT. Other factors (e.g., pancreatic-, stomach-, liver- , kidney cancers or sarcomas), including hypogonadism, hypothyroidism, and marijuana use, can cause false-positive results. Further work-up is advised before initiating treatment for TC in these cases.

Despite their limited accuracy and caveats, when measured in a standardised and timely manner and in conjunction with imaging, AFP, β-hCG and LDH still remain the cornerstone in the diagnosis, follow-up, and post-treatment monitoring of TC in the EAU Guidelines and other major national, international, or regional guidelines (e.g., NCCN, NHS, SWENOTECA, Princess Margaret Hospital, MD Anderson).

Blood circulating miRNAs When considering the above regarding the performance of the currently used biomarkers, it seems clear that new markers providing increased accuracy, and minimising false-negative or falsepositive results are desirable. Basic- and translational research is ongoing. Among other possible potential molecular biomarkers that meet the definition of a liquid biopsy, the most promising emerging candidates are blood circulating miRs.

Around 80% of the human genome is transcribed, however, only 1–3% is further translated into proteins. Most of the human genome represents non-coding RNAs (ncRNAs), which play key regulatory functions. miRs are short-ncRNAs single stranded shape molecules of approximately 19-22 nucleotides with a specific sequence. They are key molecules partially complementary to one or more messenger RNA that regulate intra- and intercellular communication in autocrine, paracrine, and endocrine pathways. miRNAs play a role in the regulation of physiological and pathological processes and are necessary for various cell mechanism such as cycle regulation, migration and invasion, drug resistance and immune response. Their dysregulation has been described in the pathogenesis of many human cancer types. miRNAs may prevent cancer transformation or promote it, in case of abnormal expression. They are stable in the blood stream but their half-life in serum is variable, mostly short.

Several clusters of miRs, including 371–373 and 302/367 but also others such as miR-367, miR-223-3, miR-449, miR-383, miR-514a-3p, miR-199a-3p and miR-214, have been proposed in several cancers as potential new biomarkers with a high specificity (> 99%). Of this group, miR-371a-3p locus, located at chromosome 19, represents the main miR cluster involved and expressed in TGCT tumorigenesis. This miR cluster is not unique to TC and regulates cell proliferation, drug resistance, migration, and invasion. However miR-371a-3p is the only miRNA extensively investigated as a TGCT biomarker in the diagnostic, staging, prognostic and follow-up setting and miR-371a-3p can discriminate between various testicular histotypes, including stromal and non-germ cell tumours.

The expression of miR-371a-3p and miR-302/367 is significantly higher in all TCGT tissue than in healthy testis and higher levels of miR-371a-3p are found in the serum of TC patients than in controls. Specifically, TGCTs derived from germ cell neoplasia in situ (GCNIS) show higher miR-371a-3p levels than non-GCNIS tumours. The expression is only slightly increased in teratomas and absent in the prepubertal and post-pubertal types of yolk sac tumours.

The higher discriminatory accuracy of miRNAs (particularly miR-371a-3p) compared to conventional TGCT markers has been confirmed in both retrospective but also prospective (multicentre) studies. Overall, depending on the clinical setting, a sensitivity and specificity of between 71% to 100% and 58% to 100%, respectively, has been reported, also depending on the clinical setting (diagnosis, early stage disease, chemotherapy response monitoring or presence of post-chemotherapy viable germ cell tumour). Areas under the curve (AUCs) were in general high although, depending on the clinical setting, could also be variable serum levels of miR-371a-3p correlate with clinical stage and tumour size and in CS I after orchiectomy, miR-371a-3p serum levels were shown to decrease by over 97% within 24 hours and over 99% in 72 hours.

At the moment, the best evidence for the M371 test (miR-371a-3p determination by quantitative polymerase chain reaction) is provided by a recent international prospective cohort study. Based on a cohort of 616 patients with either CS I or metastatic TC and 258 controls, miR levels were shown to be significantly associated with clinical stage, primary tumour size, and response to treatment. Sensitivity was shown as 90.1%, specificity 94.0%, PPV 97.2%. and NPV 82.7% with an AUC of 0.966 for the primary diagnosis of TC; all of these parameters outperforming the classical markers. The test discriminated patients with localised GCT (CS I) from those with systemic disease, with an AUC of 0.76, and a diagnostic sensitivity and specificity of 83.4% and 60.1%, respectively. miR-371a-3p levels were higher in non-seminoma and increased with higher tumour stage. Empirical data comparing the M371 test with the classic TC markers showed that the new test is significantly more sensitive than the classic markers (β-hCG sensitivity 45%, AFP 28%, LDH 32%) even when combining all three markers (60%) in all clinical settings. For each tumour stage there was a decrease in serum miR-371a-3p after chemotherapy or orchidectomy, but the miRNA-371a-3p level decreased more rapidly in earlier stages.

Characteristics of a biomarker Conventional (AFP, β-hCG) miRNAs (miR-371a-3p) Non-invasive Yes Yes Yes Easy to measure Yes Cumbersome /technical variations Highly expressed in sample Discrimination No No Yes Yes

Long-half life in biological samples Yes Inexpensive Yes

Rapid results Cost-effectiveness Yes Unknown

No Unknown Not yet generalisable Unknown Reproducibility Yes Unknown Table 1: Characteristics of classical biomarkers and miRNAs as biomarkers in testicular cancer

The data also confirmed the lack of expression of miR-371a-3p in teratoma or in case a somatic malignant component is present; further limitations of miR-371a-3p, either from this study or reported by others, include the low sensitivity (59%) in seminomas < 1 cm, the presence of false positives (up to 13% in CS I), and the lack of information regarding its role in a follow-up setting which needs to be clarified before imaging can either be replaced by this marker, or act as a reliable trigger for further imaging.

“In view of the excellent performance of miRs one can question why they are not yet included as TC biomarkers in the EAU Guidelines.“

The position of the EAU Guidelines In view of the excellent performance of miRs one can question why they are not yet included as TC biomarkers in the EAU Guidelines. When preparing the 2022 Guidelines, the TC panel once again carefully analysed and reconsidered the literature on miRNAs.

The panel acknowledge that well-designed studies present excellent results for miR -371a-3p across the TC management pathway. These results show that miR-371a-3p outperforms the current markers and may present a turning point in the management of TC, impacting all possible clinical scenarios However, whilst the potential of these biomarkers is stressed, also shortcomings in the translational pathway and possible barriers for implementation were identified, which, on balance, preclude replacing the classical serum tumour markers by miRs in the 2022 EAU Guidelines.

Usually, the process between biomarker discovery and meaningful clinical application is arduous and slow. The apparent lack of support of miRs in the TC Guidelines does not nullify the current scientific evidence but rather encourages well-designed studies with larger cohorts and appropriate follow-up for all possible clinical scenarios. Several of these projects are already ongoing on both sides of the Atlantic.

From a clinical perspective, the biochemical properties of the miRNAs in general and of the miR-371a-3p cluster in particular makes them excellent biomarkers in TC. So far, the translational work has shown that they are able to provide reliable and accurate information outperforming the present TC serum markers. However, some outstanding queries need resolving before they can be incorporated into standard clinical practice, namely from sample collection to laboratory standardization, external diagnostic and prognostic validation, determine their value in a follow-up setting, cost-efficiency, either financial or in terms of measurable patient risk and lastly how to ensure the availability of the test at the point of care.

Quality control is of paramount importance in order to avoid pitfalls that include sample collection, handling and storage, avoidance of haemolysis in the sample, extraction, and normalization. Although mainly RT-qPCR-based assays have been used, the best quantification method has yet to be determined. Overall, the technique is still time-consuming and requires sophisticated and expensive equipment which is not always available. So far results are available for miRNA 371a-3p alone, although other clusters with a longer serum half-life are already being explored. Either alone, or in combined clusters, panels may improve the prognostic ability or even expanding the diagnostic capabilities.