Study population and study design

All males aged ≥15 years in Denmark diagnosed with incident CS I TGCC between 1 January 2013 and 31 December 2018 are included in a nationwide, population-based study divided into two separate cohorts:

Study cohort I: patients with CS I pure seminoma.

Study cohort II: patients with CS I non-seminoma.

Relapse is defined as either a confirmed tumour marker relapse (hCG, AFP) and/or radiological signs of relapse. The time point for relapse can be either increase in tumour marker or a positive CT scan depending on which comes first.

End of follow-up is planned on 31 December 2021 ensuring a minimum follow-up period of 3 years of all patients. For seminoma, 90% of the relapses occur within 3 years of follow-up,3 for non-seminoma, more than 90% of relapses occur within 2 years of follow-up.1

Staging and follow-up 

All patients with TGCC are treated with inguinal orchiectomy followed by staging with STMs (AFP, hCG and LDH) and a CT scan of thorax and abdomen. CS I disease is defined as normal postoperative STMs, and no radiologic or clinical evidence of regional or distant metastatic disease. The orchiectomy procedures are carried out at local urological departments throughout Denmark with subsequent pathological examinations at 11 different pathological departments. Follow-up and treatment are harmonised in national multidisciplinary guidelines and carried out at three university hospitals responsible for this patient group. Patients with stage I disease are, regardless of pathological characteristics (pT1-4N0M0S0), followed by a uniform, national 5-year surveillance programme.1 3 After 5 years of follow-up, survival and possible relapse can be followed through national registries, as outlined below.

Data sources

Data are obtained from the prospective Danish Testicular Cancer (DaTeCa) database32 and linkage of the DaTeCa database to the following national registries: the Danish Pathology Registry (DPR),33 34 the Danish National Patient Registry (DNPR)35 and the Danish Civil Registration System (CRS),36 as specified in table 1. Individual-level linkage of data is possible due to a unique civil personal 10-digit registration number (Danish civil registration number, CPR), assigned to all Danish citizens at birth or immigration, which is recorded along with administrative and medical information in registries and databases.36 37

The prospective DaTeCa database

The nationwide clinical database holds information of all incident germ cell cancers (GCCs) in males aged ≥15 years of both gonadal and extragonadal origin in Denmark from 2013 onward.32 The database contains prospectively collected clinical data registered by the treating physicians at the oncological departments by using standardised case report forms, as previously described,32 including information on clinical stage, relapse data and preorchiectomy values of STMs (AFP, hCG and LDH) used for this study. The database is linked to the DPR and the DNPR to ensure completeness of the identification of all incident TGCC cases as well as of the reporting to the database, table 1. In total, 98%–100% of all newly diagnosed patients identified in the DPR and DNPR have an online registration form filled out during the study period 2013–2018.38 The high completeness is possible as patients identified in the registries but not registered from the departments can be localised and enquiries sent to the departments. Further, safeguarding against missing information on critical data, such as relapse, is also ensured by the crosschecking, table 1.

The Danish Pathology Registry

The DPR receives data from the Danish Pathology Data Bank (DPDB) and holds information on all pathological specimens analysed in Denmark since 1997.33 34 All Danish pathology departments have electronically recorded standard data on biological specimens according to national guidelines for uniform registration. Registration is performed by the investigating pathologist and is based on the Danish version of the Systemised Nomenclature of Medicine (SNOMED) codes.33

The Danish National Patient Registry

In the DNPR, all hospital contacts, including outpatient visits, are registered.35 Information regarding hospital admission, such as date of admission and discharge and diagnosis codes at discharge, classified according to International Classification of Disease, tenth revision (ICD-10), are registered.35 In addition, procedure codes such as chemotherapy and radiotherapy codes are registered according to the Healthcare Classification System (Danish, Sundhedsvæsenets Klassifikations System).39 40

The Danish CRS

The CRS holds information on vital status and migration which is updated daily.36

Data construction and collection

Figure 1 summarises the process of defining the cohort and collection of the orchiectomy specimens. All patients with CS I disease identified in the prospective DaTeCa database will initially be coupled to the Danish ‘Vævsanvendelsesregister’/Register of Human Tissue Utilisation,34 and patients who have registered that their tissue cannot be used for scientific purposes will be excluded. Patients with GCC of extragonadal origin, synchronous or metachronous TGCC are also excluded. By linkage to the DPDB, the identification and location of the archived histopathological slides (H&E stained slides (HE) and immunohistochemical (IHC) stained slides) from the orchiectomy specimens in the various pathology departments throughout Denmark are obtained, as well as information from the original pathology reports concerning gross and microscopic descriptions and diagnosis. The length of time for which the glass slides from the specimens are stored in the archives varies by pathology department (usually between 5 and 10 years). If any slides are discarded or missing, the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks will be collected and new HE slides will be made. As Danish pathology departments store paraffin blocks permanently,33 we expect to be able to retrieve all FFPE blocks from all the patients, if relevant. All slides will be scanned into digital images.

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Figure 1

Flowchart outlining the definition of the study population and data collection. CS I, clinical stage I; GCC, germ cell cancer; TGCC, testicular germ cell cancer.

Data analyses

Microscopic slides from the orchiectomy specimens and information from the original pathology reports will be reviewed by the same pathologist (TW) without knowledge of the clinical outcome. The most recent recommendations from the International Society of Urological Pathologists (ISUP)41 and the International Collaboration on Cancer Reporting (ICCR)42 regarding the reporting of microscopic features in testicular germ cell tumours with clear definitions of various parameters will be used. The following gross and microscopic parameters will be recorded, as defined in table 2: tumour size, tumour necrosis, LVI and tumour involvement of tunica albuginea, tunica vaginalis, rete testis, hilar soft tissue, epididymis and spermatic cord. If present in non-seminoma tumours, the histologic tumour type showing the stated feature will be recorded. Further, in non-seminoma tumours, the presence and absence of each histologic tumour type with corresponding percentages as a continued variable will be recorded. All cases will be reclassified in accordance with the WHO 2016 histological classification.43 In case of diagnostic uncertainty, the slides will be reviewed by two additional pathologists (BGT and DB) to reach a consensus. If during the review it is deemed necessary to perform IHC staining (OCT3/4, CD30, CD117, D240, Glypican3, AFP, hCG, CD31 and CD34 according to the ISUP recommendations)44 or molecular testing for chromosome 12 p amplification by fluorescence in situ hybridisation (FISH) and/or PCR, for differential diagnostic purposes, the relevant tissue block will be collected. After completing the revision, the pathological features will be tested with regard to clinical outcome (see the ‘Statistical analysis’ section). National guidelines on handling and sampling of orchiectomy specimens have been standardised in Denmark for many years,45 and is in accordance with international recommendations.42 As such, adequate sampling and thereby a minimum of missing values is expected.

Statistical analysis

The aim of this study is to investigate the association of clinical–pathological covariates as explanatory variables to the primary outcome, that is, time to relapse. Patients without a relapse will be censored at the final date or the date of death or emigration if occurring prior to the final date. The explanatory covariates are listed in table 2. Descriptive statistics will be presented in tables for categorical variables and by the median with minimum, maximum as well as first and third quartiles for continuous variables stratified by seminoma/non-seminoma. The primary analysis will be performed using the Cox proportional hazards model, analysed separately for seminoma and non-seminoma. Explanatory covariates will be scored as categorical variables if discrete, and as continuous variable if appropriate. For the latter, possible transformation will be utilised to achieve the correct functional form, the most likely transformation is the log of the explanatory covariate. Model assessment will be done employing martingale residuals to confirm the proportional hazards assumption and the functional form if applicable. If the assumption of proportional hazards is violated alternative parameterisation will be done using time-dependent Cox models. In the event of missing explanatory variables, multiple imputation will be performed with 25 imputations.46 47 Significant combinations of explanatory covariates will be identified using backwards selection validated using 10-fold cross validation.48 The results will be presented as estimates of hazard ratio (HR) with 95% confidence limits and cumulative incidence rates using the Nelson-Aalen estimator. The plausible risk factors for relapse: scrotum invasion and tumour in the spermatic cord margin, will not be included in the primary analyses in case of (and as expected) few events (<10); neither will tunica vaginalis invasion in case of few events (<10). Statistical calculations will be done using SAS and R, The R Foundation for Statistical Computing Platform.

Sample size and power considerations

Study cohort 1: With approximately 160 incident cases of CS I seminoma per year in Denmark,40 the cohort is estimated to constitute approximately 960 patients and 185 incident relapses. Study cohort II: With approximately 80 incident cases of CS I non-seminoma per year in Denmark,40 the cohort is estimated to constitute approximately 480 patients and 150 incident relapses. Assuming a 5% significance level (two sided), there will be 80% power to detect a HR of a continuous covariate of 1.21 or greater (or 0.83 or lower) in a multivariable Cox regression model with r²=0.2 regressing the covariate of interest on the remaining covariates and a SD=1.2 in study cohort 1, and 1.24 (or 0.81) in study cohort 2.49 For a binary covariate, the estimated power to detect a HR greater than 1.6 (or 0.63 or lower) is 80% if the proportion of positives is 35% in study cohort 1. If the proportion of positives is 15%, then there is 80% power to detect a HR of 1.9. Similarly for study cohort 2, the HR that can be detected at 80% power are 1.7 and 2.0.50 All analyses assume that r²=0.2 between the covariate of interest and other explanatory covariates. Most clinically, relevant HR will likely be higher, and therefore this study has sufficient power to address the research questions posed. Calculations have been performed using ‘Proc Power’, SAS V.9.4 and package ‘powerSurvEpi’, R V.3.5.0.

Patients and public involvement 

Involvement of all Danish CS I TGCC patients will be ensured through nationwide registries. Since the material used for evaluating pathological risk factor consists of slides for microscopy originally collected for diagnostic purposes, the patients are unable to participate and engage directly in the current study. Our data material is ideal for performing prognostic factor research in patients with CS I disease, and our aim is to clarify risk factors with significant influence on relapse. Thus, future CS I TGCC patients can make their decision about continued surveillance or adjuvant chemotherapy based on solid data. Potentially, our results will entail individual follow-up programmes based on risk of recurrence, which for some patients could imply less intense follow-up than we do today.