Testicular cancer is the first solid tumor with a remarkably high cure rate. This success was only made possible through collaborative efforts of basic and clinical research. Most patients with distant metastases can be cured. However, the majority of these patients are diagnosed at a young age, leaving many decades for the development of treatment-related complications. This has magnified the importance of research into survivorship issues after exposure to platinum-based chemotherapy. This research, along with research into newer biomarkers that will aid in the diagnosis and surveillance of patients and survivors of testicular cancer, will continue to advance the field and provide new opportunities for these patients. There also remains the need for further therapeutic options for patients who unfortunately do not respond to standard treatment regimens and ultimately die from this disease, including a cohort of patients with late relapses and platinum-refractory disease. Here we discuss the advancements in management that led to a highly curable malignancy, while highlighting difficult situations still left to solve as well as emerging research into novel biomarkers.
The achievements in the management of testicular cancer over the past 60 years continue to serve as a model of success of collaborative research. The diagnosis of metastatic testicular cancer once carried an almost certain fatal outcome. Today, advancements in basic and translational sciences have paved the way for a 5-year relative survival of 95% for all patients diagnosed with testicular cancer and a 5-year relative survival of over 70% for patients diagnosed with distant metastases (1). While the discovery of cisplatin in the 1960s remains the crucial advancement responsible for these unprecedented results, research into various dosing strategies, including high-dose chemotherapy, and research into biomarkers for diagnosis and surveillance, have and continue to contribute immensely to the field. These advancements have allowed researchers to expand investigative emphasis to other aspects of patient care, most notably survivorship issues. The success in management of the disease has also underscored the challenging cases that still remain, including patients with late relapses and incurable platinum refractory disease. This review aims to highlight the major discoveries and advancements that have translated into one of the greatest success stories in solid tumor oncology, while acknowledging challenges that remain, and emphasizing future directions in research.
Although the incidence of testicular cancer has been rising since 1975, it remains a rare disease, representing 0.5% of all new cancer cases in the United States in 2020 (1). It is estimated that 0.4% of men will be diagnosed with testicular cancer during their lifetime (1). Despite this, it remains the most common malignancy in males between the ages of 15–35 years old, representing a population with a high number of productive years of life still left at time of diagnosis. Germ cell tumors (GCT) make up the majority of testicular cancers. Along with their rarity in prevalence, GCTs have some biologic features that distinguish them from other typical solid tumors, most notably their histologic heterogeneity (2). GCTs are divided into seminoma and nonseminoma, each accounting for about 50% of cases. Seminomas are homogenous tumors of embryonic germ cells, while nonseminomas contain one or more histologic subtypes including embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma (3).
In the early 1970s, the discovery of a precursor lesion, now termed germ cell neoplasia in situ (GCNIS), paved the way for the beginning of the understanding of the biology and progression of GCT (2, 4). It is thought that GCNIS cells derive from gonocytes that have failed to mature. Once exposed to hormonal changes at puberty, these gonocytes grow and acquire the features of GCNIS (2, 5). Researchers have investigated possible molecular and genetic changes that may lead to the acquisition of these features. More than 80% of GCTs harbor an isochromosome of the short arm of chromosome 12, and this is a pathognomonic marker in GCT (6). Even GCTs that lack this isochromosome still have amplification of 12p genetic material. Genes localized to the 12p region are associated with pluripotency, such as NANOG, STELLAR, DPPA-5, and GDF3, and with germ cell proliferation and survival, such as CCND2 and K-RAS (6–10). Karyotyping of GCNIS cells when in the presence of invasive GCT demonstrate the i(12p), though interestingly, this isochromosome is not always found if invasive tumor is not nearby (2, 7, 11, 12). Thus, GCNIS appears to be a precursor to germ cell tumors, but critical steps occur that lead to malignant transformation of these cells, with a gain of 12p sequences possibly playing a role (13). Genome-wide association studies have aimed to identify loci that may be associated with GCT. A meta-analysis in 2017 combined five published genome-wide association studies to identify eight new GCT susceptibility loci and 19 SNPs that met genome wide significance. These loci contain genes that are involved in male germ cell development and pluripotency, including TFCP2L1 and ZFP42, DNA damage response (TIPIN), and others, thus increasing the number of known GCT susceptibility alleles to 40 (14).
Well before the knowledge of the transformation process of gonocytes into GCTs was known, chemotherapies aimed at treating patients with advanced testicular cancer were ongoing. The first of these was led by Li and colleagues in 1960, in which 36 patients with GCT received a combination of an alkylating agent, an antimetabolite, and actinomycin D. Twenty three of the patients received a full three-drug regimen, with 12 showing objective clinical and laboratory response with a duration of 1–18 months. Complete or near complete disappearance of tumors was seen in 7 of those 12 patients (15). Further studies demonstrated similar findings, with an objective response rate of around 50% for actinomycin D–based therapies (16). Mackenzie and colleagues in 1966 treated 154 patients with metastatic GCT with actinomycin D alone or in combination and noted 24 of the patients were without any evidence of disease, with half of those maintaining that response and thus essentially cured (16). With the introduction of bleomycin in the 1970s, studies involving further combinations of bleomycin with vinblastine began. Vinblastine disrupts the mitotic spindle, thus arresting cells in mitosis. Early studies of bleomycin in vitro noted that it was most effective in killing hamster ovary cells in mitosis, thus the combination of bleomycin with vinblastine demonstrated a synergism not seen in previous chemotherapy combinations, with 57% of patients with testicular cancer achieving a complete response (CR) and 45% of them remaining without evidence of disease (17, 18).
Cis-diamminedichloroplatinum, or cisplatin, was first shown to have antibiotic activity in the mid 1960s, inhibiting the growth of E. coli (19). The first demonstration of the antitumor effects of cisplatin occurred in mice with leukemias and sarcomas, and it was approved for use in clinical trials by the NCI in 1972 (20–22). Several studies of cisplatin as a single agent in metastatic testicular cancer did show activity, with an overall response rate around 55%, though it was not until cisplatin was combined with other chemotherapies for multi-drug combinations that its full potential was revealed (23, 24). In 1974, 47 patients with metastatic testicular cancer were treated with the three-drug regimen of cisplatin, vinblastine, and bleomycin (PVB). Thirty three patients (70%) achieved a CR, and 27 (57%) of them were alive without evidence of disease at 5 years (25). Etoposide entered clinical trials shortly after and showed efficacy as a single agent in patients with refractory GCT (26). In 1984, the three-drug regimen of PVB was compared with a combination of bleomycin, etoposide, and cisplatin (BEP) at Indiana University (Indianapolis, IN) in 244 patients with metastatic GCT (26). The CR rate in the BEP group was 78% compared with 66% in the PVB group (P < 0.05), thus BEP became and remains standard of care for patients with metastatic testicular cancer (27). In patients with underlying pulmonary disease, etoposide, ifosfamide, and cisplatin (VIP) is an alternative, though is associated with slightly higher hematologic toxicity (28). Four cycles of etoposide and cisplatin (EP) remains an option for good-risk disease, with a more recently updated study of 944 men with good-risk GCT treated with this regimen showing a 5-year progression-free survival of 93.9% and 5-year overall survival of 97.9% (29).
In 1997, the International Germ Cell Cancer Collaborative Group (IGCCCG) released a classification system that divided patients into risk groups based on several clinical factors (30). This system was recently updated, with the addition of other adverse prognostic factors. Most notably, though, there were marked improvements in survival outcomes for all risk groups as compared with 1997, likely the success of the standard-of-care chemotherapy now used (31, 32). Close to 90% of patients with good risk disease achieve cure with chemotherapy, while just 50%–60% of those with poor risk disease are cured (33). These initial studies resulting in curing an adult solid tumor were a remarkable achievement. Even more impressive was offering curative potential to patients with second- or third-line salvage chemotherapy (34). Between 1986 and 1988, 33 patients with refractory GCT were enrolled in a phase I/II trial of two courses of high-dose carboplatin plus etoposide followed by autologous bone marrow transplant (35). Sixty-seven percent of these patients were platinum refractory, indicating they had progressed within 4 weeks of the last platinum dose, and 35% had progressed after two previous cisplatin-containing regimens. Overall response rate was 44%, with 8 patients (24%) obtaining a CR despite such advanced disease. In 1996, peripheral blood stem cells replaced bone marrow transplantation. In 2007, Einhorn and colleagues reported a retrospective review of 184 patients with relapsed metastatic testicular cancer treated with high-dose chemotherapy (HDCT) with carboplatin and etoposide followed by autologous stem cell transplantation (ASCT; ref. 36). Of the 184 patients, 116 (63%) achieved a complete remission without relapse at a median follow-up of 48 months. One hundred thirty five patients received HDCT as second line, with 94 (69%) of them remaining disease free. Perhaps, the most striking is that 22 of 49 patients (45%) who received treatment as third line were disease free at follow-up. Eighteen of 40 patients (45%) with platinum refractory disease also remained disease free. In 2017, a larger retrospective analysis from Indiana University's experience with HDCT and ASCT revealed a 2-year-progression-free survival of 60% in 364 patients with metastatic GCT, again demonstrating cures in patients treated with second- and even third-line therapy (37). Figure 1 demonstrates a timeline of important discoveries and landmarks in the history of testicular cancer.
Conventional-dose salvage chemotherapy remains an option for patients who initially relapse after first-line chemotherapy. Second-line salvage therapies include paclitaxel, ifosfamide, and cisplatin (TIP) or vinblastine, ifosfamide, and cisplatin (VeIP), with CR rates as high as 65% at 5 years as seen in the TIP regimen (38, 39). An ongoing trial (NCT02375204) evaluating conventional-dose salvage chemotherapy versus HDCT with ASCT as initial salvage treatment in relapsed GCT is under way (40). Despite salvage therapy, there remains a subset of patients who will not be cured. Treatment options for these patients remain challenging. Combination gemcitabine and oxaliplatin or gemcitabine and paclitaxel are options, with an overall response rate around 21%–46% (41, 42). In 184 patients treated with HDCT, 32 patients progressed and were subsequently treated with gemcitabine and paclitaxel. Ten patients achieved a CR, with 4 maintained NED at >20 months (43). Thus, a very small fraction of patients are able to achieve long-term disease-free survival with even post-HDCT salvage therapy.
With the success in treatment of testicular cancer, researchers have been able to move from focusing primarily on treatment options and now toward maintaining quality of life and minimizing consequences of treatment for patients. Because the median age of diagnosis is 33 years, a large amount of productive years of life remain for these patients, thus minimizing toxicities of therapy becomes crucial. Even 10–20 years later, plasma platinum concentrations are significantly higher in patients with GCT who received platinum-based chemotherapy compared with patients with GCT never exposed to platinum (44). Testicular cancer survivors are faced with a number of issues including cisplatin induced ototoxicity, neuropathy, renal toxicity, cardiovascular toxicity, metabolic syndrome, hypogonadism, infertility, and secondary malignancies. In a large study evaluating nontesticular cancer mortality in over 5,000 men diagnosed with testicular cancer between 1980 and 2009, the most important cause of death was nontesticular second cancer. Rates were even higher in patients who had received radiotherapy (45). These results were similar to other findings of increased risk of secondary malignancies, particularly in patients who received more than one line of therapy (46).
Since the 1980s, the effects of cisplatin on hearing loss were well documented, including the fact that the probability of hearing loss was correlated to the cumulative dose of cisplatin (47). In 2016, 488 men were evaluated as part of an NCI funded study evaluating late complications of cisplatin combination chemotherapy (48). Pure-tone air conduction thresholds were obtained for patients at frequencies of 0.25–12 kHz, which covered the speech frequency range as had been found in previous studies. Cumulative cisplatin dose was compared with the air conduction threshold at each frequency in the 0.25–12 kHz range. A patient's overall hearing status across all frequencies was then derived by calculating the geometric mean of air conduction hearing thresholds, and this was compared with age-specific normative samples. Researchers found that 80% of patients experienced hearing loss, with mild, moderate, moderately severe, and severe/profound hearing loss in 25%, 16%, 21%, and 18% respectively, as determined by the American Speech-Language-Hearing Association (ASHA) criteria. Increasing cumulative cisplatin doses were associated with worse hearing outcomes, with cumulative cisplatin doses of >300 mg/m2 associated with increased ASHA severity classes. After adjusting for both age and time since chemotherapy, it was noted that for every 100 mg/m2 increase in cumulative cisplatin dose, a 3.3 dB decline in overall hearing threshold occurred. One in 5 patients suffered hearing loss to the level at which hearing aids are typically recommended, though utilization of hearing aid devices is considerably less than 20%.
In 2018, Wheeler and colleagues sought to identify genetic variants that might increase the risk of cisplatin-associated ototoxicity (49). Peripheral blood was taken from 511 GCT survivors for a genome-wide association study with over 5 million common SNPs using quantitative audiometry. The investigators identified a single SNP, rs62283056, located in the first intron of WFS1, which encodes wolframin ER transmembrane glycoprotein, that met genome-wide significance for an association with cisplatin-associated ototoxicity. Mutations in WFS1 had already been described in other hearing loss conditions, making this a plausible gene to be involved in cisplatin-associated toxicity (50). WFS1 is responsible for encoding wolframin, a protein involved in controlling the endoplasmic reticulum (ER) stress response through degradation of a transcription factor involved in ER stress signaling, known as ATF6a (51). Both dysfunction of wolframin through WFS1 loss-of-function and cisplatin induce ER stress and lead to apoptosis, suggesting that the genetic mechanisms for hearing loss in cisplatin-associated ototoxicity is similar to general hearing loss patterns (49, 52). Genome-wide association studies of other cisplatin-associated toxicities, have revealed genetic variations in FAM20C as a risk factor for the development of cisplatin-associated neurotoxicity (53).
Initial studies of the prevalence of hypogonadism in testicular cancer survivors estimated an overall incidence of 11%–16%, with higher rates in those treated with platinum-based chemotherapy compared with surgery alone (54). In a study of 199 patients with GCT, symptomatic hypogonadism requiring testosterone replacement was observed in 11% of patients who had received surgery plus chemotherapy, though 5% of those who received surgery alone were still found to have symptomatic hypogonadism (55). The overall prevalence of biochemical hypogonadism, defined as a serum testosterone <300 ng/dL, was 48% with mean testosterone levels lower in the surgery plus chemotherapy group, suggesting a high proportion of asymptomatic hypogonadism (55, 56). Our current recommendation is to reserve testosterone replacement therapy for men with symptomatic hypogonadism.
GCT survivors have an estimated 7-fold increased long-term risk for cardiovascular disease compared with those without a history of GCT (57–59). Metabolic syndrome is a major risk factor for cardiovascular disease and is a condition consisting of insulin resistance, hypertension, hypertriglyceridemia, low high-density lipoprotein cholesterol levels, and obesity (60). There initially was conflicting evidence in regards to the risk of metabolic syndrome in survivors of testicular cancer (61, 62). Abu Zaid and colleagues reported the first and largest North American testicular cancer survivor cohort and examined metabolic syndrome and associated risk factors in these patients (59). In contrast to some of the previously discussed survivorship issues, the prevalence of metabolic syndrome did not differ by cumulative dose of cisplatin. Survivors with metabolic syndrome had higher rates of obesity, hypogonadism, and elevated soluble intercellular adhesion molecule-1 (sICAM) levels, which is a known marker in cardiovascular disease, as compared with survivors without metabolic syndrome. As compared with controls, testicular cancer survivors in general had similar rates of metabolic syndrome (21% vs. 22.4%; P = 0.59), though survivors were more likely to have hypertension (43.2% vs. 30.7%; P < 0.001), elevated low-density lipoprotein cholesterol ≥160 mg/dL (17.7% vs. 9.3%; P < 0.001), total cholesterol ≥240 mg/dL (26.3% vs. 11.1%; P < 0.001), and body mass index ≥25 kg/m2 (75.1% vs. 69.1%; P = 0.04) even though they were statistically significantly more likely to participate in moderate (93.8% vs. 42.4%; P < 0.001) to vigorous (66.7% vs. 33.5%; P < 0.001) intensity physical activity and less likely to be current smokers (9.3% vs. 25.9%; P < 0.001). It was postulated that cancer-associated metabolic syndrome likely has different etiologies than metabolic syndrome in the general population, with hypogonadism and chemotherapy likely being the driving forces in metabolic syndrome in testicular cancer survivors (59, 63).
Remaining challenges: a note on platinum refractory disease, late relapses, and primitive neuroectodermal tumor
Although the majority of young males diagnosed with testicular cancer will now be cured of their disease, several challenges remain. About 10%–20% of patients with testicular cancer who initially achieve a CR will relapse. Those who relapse within 4 weeks of platinum-based chemotherapy are referred to as having platinum refractory disease. Salvage therapy with surgery, standard-dose chemotherapy, or HDCT with ASCT will still cure a proportion of these men, but those patients who have failed multiple chemotherapy regimens have exceptionally low cure rates. To date, novel, noncytotoxic treatment approaches with immunotherapy or other targeted therapy have failed to produce meaningful benefit in this refractory patient population (64–71).
Genomic analysis of patients with relapsed refractory disease may help identify the mechanism behind platinum resistance and aid with more targeted therapeutics. Bagrodia and colleagues sought to identify genetic alterations that were associated with cisplatin resistance in GCT using whole-exome sequencing of 19 tumors from men with cisplatin resistance followed by next-generation sequencing of these tumors and 161 additional patients with advanced GCT (72). The overall rate of mutations per megabase for the 19 tumors was low at just 0.9, perhaps indicating why immunotherapy is less effective in GCT. Two patients with cisplatin resistant disease were noted to have alterations of TP53, and 3 patients had amplification of MDM2, thus 5 total patients with cisplatin-resistant disease had alterations in the TP53/MDM2 pathway, while none of the cisplatin-sensitive tumors had these alterations. The specific TP53-associated gene, MYCN, was noted to be amplified in 5 patients, all of whom had cisplatin-resistant disease (72). The genomics of 49 patients with GCT were evaluated to identify features that may be associated with chemotherapy resistance (73). Again, the mutational burden was low at just 0.9 mutations per megabase. KRAS was the most frequently altered gene, with the only other significantly altered gene being RPL5, which is thought to potentially regulate the MDM2-TP53 pathway (74). Other investigations into the genetic alterations in patients with relapsed refractory GCT have revealed potentially targetable genomic alterations in EGFR, ERBB3, KIT, and MET pathways (75).
While the majority of patients that recur will relapse within 2 years of therapy, 2%–3% will experience a late relapse, or relapse occurring more than 2 years from initial therapy (76, 77). Most of these late relapses happen more than 5 years from completion of initial chemotherapy. In 1995, the largest series of patients with late relapse were evaluated to focus on the incidence, characteristics, and management of these patients (78). The primary sites of relapse in these patients were retroperitoneum and lungs. These tumors were chemosensitive, though rarely cured by chemotherapy alone, even HDCT. Surgical resection is the definitive therapy (76–78). A large proportion of patients with late relapse are asymptomatic, with their only marker of disease being an elevated AFP (77). This emphasizes the importance of routine follow-up with serum tumor markers in patients more than 5 years from chemotherapy, as this increases the probability of detecting late relapse when localized and surgically curable.
Another challenging clinical situation is malignant transformation of teratoma to primitive neuroectodermal tumor (PNET). Teratoma is a pluripotent tissue that can transform along ectodermal, endodermal, or most commonly mesodermal lines. Transformation of teratoma to PNET results from transformation along the mesodermal lines (79, 80). PNET tumors are classified into central and peripheral, with central usually occurring in the central nervous system and peripheral classified as part of the Ewing family of tumors (81, 82). PNET in GCT usually is classified as central PNET, though they lack the typical translocations of chromosome 22 and instead harbor the isochromosome i(12p) seen in GCT (82). Despite originating from GCT, PNET is resistant to cisplatin-based chemotherapy, thus regimens typically used for Ewing family of sarcomas are instead utilized (80, 83, 84). PNET and other small round blue cell tumors can be seen in the orchiectomy specimen or post-chemotherapy RPLND pathology. This pathology should be an indication for PNET-specific postoperative adjuvant chemotherapy. In a series of 86 patients with PNET transformed from testicular teratoma, surgical resection was the preferred therapy if feasible, and 68 patients underwent surgical resection alone (85). Eighteen patients with metastatic PNET were treated with PNET-specific therapy of alternating cyclophosphamide, doxorubicin, and vincristine (CAV) with ifosfamide and etoposide (IE). Among 12 patients with unresectable disease, 9 had a partial response. Six patients had surgical resection followed by adjuvant CAV/IEx4 cycles and all 6 remained disease free at most recent follow-up. Therefore, adjuvant CAV/IE is recommended for patients undergoing curative resection of PNET and for those with unresectable, metastatic disease.
Biomarkers: Current State and Future Directions
In the late 1970s, shortly after the introduction of cisplatin-based chemotherapy for GCT, β-human chorionic gonadotropin (β-hCG), alpha-fetoprotein (AFP) and lactate dehydrogenase began to be used as tumor markers for testicular cancer. These tumor markers quickly became integrated in the treatment paradigm including diagnosis, staging, response assessment, and detection of recurrence (86). β-hCG is a glycoprotein secreted by syncytiotrophoblastic cells of the normal placenta, while AFP is the major protein found in the human fetus (86). Both are elevated in nonseminomatous GCT, while AFP is never elevated in seminomatous GCT. LDH is a nonspecific tumor marker for GCT and tends to be an estimation of tumor burden (86). In our opinion, it should never be used as a sole criteria for risk stratification or initiation of chemotherapy. While these tumor markers can aid in detecting cancer that otherwise is not yet clinically perceptible, their sensitivity and specificity remain relatively low. At presentation, AFP is elevated in 26%–34% of all patients with GCT, β-hCG is elevated in 38%–47% of cases, and LDH is elevated in 33%–44% of cases, with these figures being dependent on histology and stage of disease (87–89). Elevations of AFP also occur in hepatocellular carcinoma, pancreatic cancer, gastric cancer, cirrhosis, viral hepatitis, and trauma (90, 91). β-hCG has some cross-reactivity with the β subunit of luteinizing hormone (LH), resulting in a false-positive result in situations with elevated LH, most notably hypogonadism (92). Marijuana use can also falsely elevate β-hCG levels (93).
An emerging knowledge of research into small noncoding RNAs (sncRNA) began in the early 2000s. A subset of these sncRNAs, known as miRNAs appear to affect cell growth, differentiation, and apoptosis (94, 95). Serum miRNAs are elevated in all GCT, regardless of age, site, or histology (96, 97). Two clusters in particular, miR-371–373 and miR-302, are overexpressed in all malignant GCT (97). Early studies evaluating their use in patients with GCT revealed high sensitivity and specificity (98).
Dieckmann and colleagues evaluated 616 patients with GCT and 258 control patients and found that patients with GCT could be distinguished from controls based on miR-371a-3p levels with a sensitivity of 90.1% and a specificity of 94%, higher than all three conventional tumor markers combined (97). Positive predictive value (PPV) was 97.2% and negative predictive value (NPV) was 82.7%. Serum levels of miR-371a-3p also correlated with tumor size and disease stage, with sensitivities of 86.7%–98.4% for detection of even low clinical stage disease, a particularly important limitation of conventional tumor markers. Levels of miR-371a-3p appropriately decreased in response to therapy and rose with relapse, with an estimated 82.6% sensitivity to detect relapse. More recent investigations of miR371 expression confirmed these results, demonstrating a specificity of 100%, sensitivity of 96%, PPV of 100%, and NPV of 98% in 111 patients with GCT (99). One major limitation of miR-371a-3p is the lack of expression in teratoma, which limits clinical utility in certain clinical scenarios (99). Despite these encouraging results, in our experience, we have experienced a higher incidence of false-negative miRNA-371.
The advances in the management of testicular cancer remain one of the greatest successes in modern medicine. These discoveries over the past half century have led to the first metastatic solid tumor to have a cure rate >70%. With such success, researchers have now been able to focus attention to improving quality of life in survivors of GCT, as well as focusing on challenging cases that still unfortunately claim the lives of men with testicular cancer. It is only with a continued collaborative research that major breakthroughs will continue to occur and continue to aid in the diagnosis, management, and surveillance of patients and survivors of testicular cancer.
No disclosures were reported.