Novel preclinical models, therapies and biomarkers for testicular cancer
Rosas Plaza, Fernanda
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10.33612/diss.119056452
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Rosas Plaza, F. (2020). Novel preclinical models, therapies and biomarkers for testicular cancer. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.119056452
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Chapter 6
Summary, general discussion and future perspectives
SUMMARY
Testicular cancer (TC) is the paradigm of drug sensitive solid tumor types. Despite the fact TC is a highly heterogeneous disease, TC patients with metastatic disease in general respond well to cisplatin-based chemotherapy, irrespective of tumor subtype and histology. The vast majority of metastatic TC patients can be cured with standard treatment1–4. However, there is a group of TC patients
who are refractory to systemic therapy and ultimately will die of the disease. Unfortunately, clinical trials are difficult to perform because of the rarity of TC and high overall survival rates of TC patients. Therefore, new therapeutic options for TC patients who do not respond to standard chemotherapy are hardly ever explored.
First-line treatment for metastatic disease consists of cisplatin-based chemotherapy5. In 1975, the introduction of combined vinblastine and etoposide
therapy improved the cure rate of metastatic patients from 10% to 33%. Nonetheless, the real breakthrough occurred when cisplatin was evaluated shortly after in phase 1 and 2 trials. The phase 2 trial that evaluated the combination of cisplatin with vinblastine and bleomycin resulted in 74% complete remissions and a 5-year survival of 64%6, 7. Since the initial study of Einhorn and colleagues,
cisplatin is considered the corner-stone of TC standard combination treatment. The introduction of etoposide and refinement of the dosing regimens led to the extraordinary response rate of metastatic TC to chemotherapy. Later on, a phase 3 trial compared the efficacy of the BEP regimen (bleomycin, etoposide, cisplatin) versus PVB chemotherapy (cisplatin, vinblastine and bleomycin) showing superior efficacy and less toxicity with BEP regimen8. The combination
of cisplatin, etoposide and bleomycin cures around 80% of TC patients with metastatic disease9, which implies that 20% of the TC patients with metastatic
disease will need salvage treatment. Presence of relapse or refractory disease after first-line chemotherapy results in a 2-year survival of 60% with second-line treatments10. In refractory disease, defined as not normalizing tumor markers
during chemotherapy treatment or progressive disease within 4 weeks after chemotherapy completion, the 2-year survival rate is even lower (33%)10.
It is crucial not only to find novel therapies for patients who do not respond to chemotherapy, but also to identify the population that has a higher chance of developing relapse or refractory disease before start of treatment. The International Germ Cell Cancer Consensus provided a classification that stratifies patients into good, intermediate and poor prognosis groups based on several clinical features. Importantly, the poor prognosis group has a 48% 5-year overall survival11. Accurate prediction of adverse outcomes should preferably
happen prior to treatment, or early in the course of therapy in order to have the possibility to intensify or change the chemotherapy regimen. Biomarkers are
currently being investigated to improve the identification of patients who are at risk of poor treatment outcome.
Little is known about the cause of cisplatin resistance in TC. Nonetheless, some of the few altered genes found in resistant TC tumors have a function within the MDM2-p53 axis as well as in mitogenic pathways. Altered genes that have been involved in cisplatin resistance are TP53, MDM2, KRAS, AKT1, MTOR and PIK3CA12, 13. In addition, hyperactivation of the PI3K/AKT/mTORC
pathway in orthotopic in vivo TC models was associated with a more aggressive phenotype3, 14. However, current preclinical knowledge of cisplatin-sensitizing
agents has not been translated to the clinic. Several targeted drugs inhibiting the PI3K/AKT/mTORC pathway have been tested in phase II trials as single agents without promising results15, 16.
In this thesis, we aimed to gain insight into the cisplatin-based chemotherapy response in TC. We used a cohort of metastatic TC patients to investigate the behavior of the putative biomarkers miR-371a-3p, miR-372-3p and miR-367-3p in response to chemotherapy. Additionally, we explored how to sensitize TC cell lines and human TC-based animal models to cisplatin. The purpose of both projects was to improve detection of patients that do not fully respond to first-line treatment and provide potential novel treatments for them. To this end, we developed and characterized patient-derived xenografts (PDX) that were used to test combinatorial therapies explored previously with cell lines in vitro. In Chapter 1, an overview of clinical and biological characteristics of TC was provided. We reviewed general aspects of diagnostic and therapeutic management of TC and we gave insight into the mutational landscape of the disease. In this chapter, we also presented information on current knowledge of cisplatin sensitivity in TC and results of clinical trials in targeted drugs to treat chemotherapy- resistant TC patients.
In Chapter 2, we investigated three microRNAs: miR-371a-3p, miR-373-3p and miR-367-3p as putative tumor biomarkers during and after treatment with cisplatin combination chemotherapy in patients with metastatic testicular germ cell cancer (TC). LDH (lactate dehydrogenase), AFP (alpha-fetoprotein) and ß-HCG (human chorionic gonadotropin) are used in diagnosis, and follow-up of TC patients. Our aim was to investigate the association between levels of miR-371a-3p, miR-373-3p and miR-367-3p and clinical features in metastatic TC. Relative levels of miR-371a-3p, miR-373-3p and miR-367-3p were evaluated in serum of metastatic TC patients. A prospectively-included and a retrospectively-selected cohort were studied (total patient number= 109). Blood samples were drawn at start of chemotherapy and during follow-up. Serum microRNA (miR) levels were determined using the ampTSmiR test. At start of chemotherapy,
miR-371a-3p, miR-373-3p and miR-367-3p levels were positively correlated to LDH. The median level of these miRs was higher in patients who developed a relapse after complete biochemical remission (n=34) than in those who had complete durable remission (n=60). Higher levels of miR-367-3p were found in patients with refractory disease (n=15) compared to those who had complete response. In patients with complete response, levels of these miRs decreased after the first week of chemotherapy and stayed below threshold after one year of treatment. In conclusion, high levels of 371a-3p, miR-373-3p and miR-367-3p at start of chemotherapy are associated with worse clinical outcome and can assist in early diagnosing of tumor relapses.
In Chapter 3, we explored the use of kinase inhibitors to sensitize testicular cancer cells to cisplatin treatment. Activation of kinases, including receptor tyrosine kinases, and downstream substrates was studied in five cisplatin-sensitive or resistant TC cell lines using phospho-kinase arrays and western blotting. The phospho-kinase array showed AKT and S6 to be among the top phosphorylated proteins in TC cells, which are part of the PI3K/AKT/mTORC pathway. Inhibitors of most active kinases in the PI3K/AKT/mTORC pathway were tested using apoptosis assays and survival assays. Two mTORC1/2 inhibitors, AZD8055 and MLN0128, strongly enhanced cisplatin-induced apoptosis in all tested TC cell lines. Inhibition of mTORC1/2 blocked phosphorylation of the mTORC downstream proteins S6 and 4E-BP1. Combined treatment with AZD8055 and cisplatin led to reduced clonogenic survival of TC cells. Two TC patient-derived xenografts (PDXs), either from a chemo-sensitive or -resistant patient, were treated with cisplatin in the absence or presence of kinase inhibitor. Combined AZD8055 and cisplatin treatment resulted in effective mTORC1/2 inhibition, increased caspase-3 activity, and enhanced tumor growth inhibition. In conclusion, we identified mTORC1/2 inhibition as an effective strategy to sensitize TC cell lines and PDX models to cisplatin treatment. Our results warrant further investigation of this combination therapy in the treatment of TC patients with high risk relapsed or refractory disease.
In Chapter 4, we described the establishment and characterization of TC PDX models. Pre-clinical models that faithfully reflect the patient tumor are needed to assist in target discovery and advanced drug development. PDX models of TC were established by subcutaneous implantation of solid tumor pieces in NOD scid gamma (NSG) mice. From 8 TC tumors that were implanted, 3 were established as a PDX model. PDX models and matched patient tumors were characterized using immunohistochemistry, showing retention of histological subtypes over several passages. Copy number variation analysis and RNA-sequencing was performed on PDX tumors to assess the effect of passaging, showing high concordance between passages. Chemosensitivity of PDX models corresponded with patients’ response to chemotherapy. In conclusion, we established and characterized 3
TC PDX models. These models faithfully reflected chemosensitivity and can now be used for mechanistic studies, therapeutic development and pre-clinical validation of novel therapeutic strategies in testicular cancer.
LDH, AFP and ß-HCG are used in diagnosis and follow-up of TC patients. However, not all tumors from TC patients secrete these proteins. Moreover, elevated LDH and AFP serum levels can be found with pathologies different from TC. In Chapter 5, we aimed to validate PDX models as suitable models to study TC biomarkers. We selected three PDX models, derived from both AFP-positive and ß-HCG-positive patients. Two models were derived from cisplatin-sensitive TC patients (TC1 and TC5) and one from a cisplatin-resistant TC patient (TC4). AFP and ß-HCG serum levels were measured using ELISA. TC1, TC4 and TC5 all produced AFP. The PDX model derived from a patient, who presented with high ß-HCG serum levels, did not secrete detectable ß-HCG levels in sera of mice. To monitor AFP levels in TC PDX mice during treatment, we analyzed serum samples at start and end of a cisplatin treatment cycle. Notably, there was an inverse relation between AFP serum levels and tumor volume or response to treatment in both PDX models. In conclusion, our results suggest that AFP and ß-HCG cannot be reliably used as tumor markers in PDX models from TC. In Chapter 6, a summary, general discussion and future perspectives were given.
DISCUSSION AND FUTURE PERSPECTIVES
Improving patient stratification and early detection of treatment failure in TC
Prognosis stratification has helped oncologists since 1997 to identify TC patients with a higher risk of dying of the disease. Metastatic patients with intermediate and poor prognosis receive 4 courses of BEP chemotherapy (bleomycin, etoposide and cisplatin), while patients with good prognosis receive only 3 courses. In addition, patients with poor prognosis have between 45-50% 5-year survival rates. This means that half of the patients with poor risk are not receiving effective cancer treatment, and might benefit from more effective strategy. There have been efforts to identify the patients with the highest risk of treatment failure, within the poor prognosis group17. Some prognostic factors
that affect overall survival (OS) of TC patients are brain metastasis, higher age and a primary mediastinal non-seminomatous tumor histology18, 19. Our results
indicate that determining miR-367-3p levels at start of chemotherapy could hold valuable information and can be added to the already known prognostic factors (Chapter 2). Moreover, we demonstrated that miR-371a-3p, miR-373-3p and miR-367-3p levels can be more sensitive than AFP, LDH or ß-HCG in detecting suboptimal tumor response to chemotherapy. Finally, we estimated half-life of miR-371a-3p in a small subset of patients (Chapter 2). Comparing miRNA half-life of patients with complete response versus patients with refractory disease or early relapse might inform us about the value of miRNA half-life determination for patient stratification already within the first week of chemotherapy. A recent study actually showed that longer miRNA half-life in poor outcome patients might indicate a more aggressive phenotype20. Studies evaluating miRNA kinetics found
a wide range of degradation dynamics, resulting in heterogeneous miRNAs half-lives21, 22. Nonetheless, it is clear that most miRNAs are usually degraded within
hours. In TC patients, miRNA levels decrease quickly after orchiectomy (<12 hours)20 and after start of chemotherapy (~27 hours)23, implying that detectable
serum levels seen in TC patients are the result of continuous production by viable tumor cells. Thus, high levels and slower decline of miRNAs might coincide with tumor burden, impaired response to chemotherapy and poor risk disease. Challenging aspects of introducing miRNA in clinical practice are mainly technical. Robust and reproducible miRNA quantification is prerequisite, with reliable equipment and a specialized team, able to measure miRNA levels and analyze them with the appropriate controls. Additionally, interpretation of miRNA serum level data is not straightforward. Therefore, clinicians should be trained to interpret the results. International standard cut-offs to define elevated levels in TC patients, might impose an extra limitation. Moreover, not all patients had detectable miRNA levels after orchiectomy; therefore, additional tumor markers need to be included to improve early detection of relapse or refractory disease.
TC PDX models may be of great help for biomarker evaluations. Disappointingly, we did not find a correlation between TC tumor volume and AFP levels in blood of mice (Chapter 5), questioning the possibility usefulness of studying these serum markers in TC PDX models. A drawback of our study was the low number of models, while not all subtypes of TC were represented as PDX models (Chapter 4). This will also limit the value of miRNA-based biomarker studies in our current TC PDX models.
Strategies to improve survival of TC patients with poor outcome
Improved patient stratification and early detection of treatment failure in TC using biomarkers as described above will only be of value when alternative treatment options can be offered. To improve survival of patients with poor outcome, regimen intensification by increasing the dose early in the course of therapy has been suggested. A phase 3 clinical trial evaluated the effect of personalized chemotherapy based on tumor marker decline24. At day 18-21 after
start of chemotherapy, tumor markers were evaluated based on an algorithm to determine unfavorable marker decline and half of the patients were randomized to receive either 3 additional cycles of BEP (standard treatment) or a dose-dense regimen. The dose-dense regimen consisted of 2 cycles of BEP/oxaliplatin/ paclitaxel followed by 2 cycles of cisplatin/bleomycin/ifosfamide. Although PFS and other clinical parameters improved in dose-dense regimen treated patients, OS was not statistically different compared to standard treatment. These results suggest that surviving tumor cells leading to an unfavorable tumor decline could stand as the subset of intrinsic chemo-resistant cells. Therefore, treating the cancer cells in these patients with a higher dose of chemotherapy is not likely to improve response considerably.
Alternative treatment strategies include PARP inhibitors or immune checkpoint inhibitors. TC cells have a reduced DNA repair capacity, and drugs affecting DDR, such as PARP inhibitors, in combination with platinum might be another therapeutic option for testicular tumors, as some studies using PARP inhibitors in TC cell lines have suggested25, 26. Currently, a phase II study with gemcitabine,
carboplatin and the PARP inhibitor veliparib (ABT-888) is performed in refractory TC patients (Clinical trial: NCT02860819). Much enthusiasm has emerged from the development of immune checkpoint inhibition (ICI) and its efficacy in treating several types of cancer. Data available so far shows that TC tumors are not responsive to ICI monotherapy27. Combined ICI treatments
have improved survival outcomes in melanoma28, and those combinations are
now studied in TC as well (NCT03333616). Unfortunately, PDX models cannot be used for testing of ICIs due to the lack of an immune system in NSG mice. Reconstitution of autologous or allogeneic human immune cells might be an option but is still a major challenge29.
Potentially actionable mutations in TC have been identified in the p53-MDM2 axis, PI3K/Akt/mTORC signaling and MAPK signaling12, 30, 31. In Chapter 3, we
examined responses of TC cell lines towards specific kinase inhibition, focusing on mTORC1 and mTORC2. Inhibition of mTORC1 has, however, proven some disadvantages for cancer therapy in preclinical and clinical studies32.
Interestingly, we found that mTORC1/2 inhibition resulted in an efficient blockade of the mTORC pathway and sensitizes TC cell lines and TC PDX models towards cisplatin without inducing too high levels of toxicity in mice (Chapter 3 and 4). These results warrant further investigation of this combination therapy in the treatment of TC patients with high risk relapsed or refractory disease. The mTORC1/2 inhibitor TAK228 (MLN0128) is currently being tested in clinical trials for advanced solid tumors33. However, it is an open question whether
cisplatin can be combined with mTORC1/2 targeted drugs for the treatment of TC. Adding TKI inhibition to cisplatin has been rarely explored in clinical trials, but epidermal growth factor receptor (EGFR) inhibition in combination with platinum-based chemotherapy significantly increased OS and PFS in patients with small cell lung cancer, particularly in a subset of patients with EGFR mutations50, indicating the feasibility of such an approach in some cases.
The link between PI3K/AKT/mTORC1/2 pathway activation and cisplatin resistance in TC tumor specimens has not been firmly established. Therefore, it is necessary to study this relation systematically by evaluating the expression of phospho-proteins, indicative of PI3K/AKT/mTORC1/2 pathway activation, in TC tumors from cisplatin-resistant and –sensitive patients. We are currently building a TC tumor specimen collection with registered clinicopathological characteristics to validate our findings obtained in TC models, and to identify putative markers of PI3K/AKT/mTORC1/2 pathway activation in TC that can be used to select patients who may benefit from mTORC1/2 targeted treatment.
Mechanisms of drug sensitization by mTORC1/2 inhibition in TC
Targeting the PI3K/Akt/mTORC pathway using TKIs or upstream inhibition of the pathway has shown limited efficacy in the clinic34, with some exceptions35.
A previous report demonstrated that cancer cells are highly capable of compensating kinase inhibition by upregulating membrane receptors36 or
inducing activation of other kinases as a consequence of inhibitory feedback loss. In line with published data37, we observed induction of AKT phosphorylation
after mTORC1 inhibition upon everolimus in resistant TC cells. Inhibition of mTORC1 and mTORC2 resulted in an efficient blockade of the mTORC pathway without upregulation of AKT-Ser473 phosphorylation, therefore hampering full activation of AKT. This phenomenon might explain the high sensitivity of all TC cell lines towards mTORC1/2 inhibition (Chapter 3). It is not known what the
driver(s) of PI3K/Akt/mTORC pathway activity are in TC. Two previous studies in a limited number of TC cell lines demonstrated that PI3K/AKT activation in these models was driven by PDGFRß and IGF1R3, 38. However, we could not
confirm these results in our models.
We observed that mTORC1/2 inhibition elevated cisplatin-induced apoptosis levels in TC cells. Involvement of mTORC1/2 in cellular processes like growth, autophagy and metabolism are well described39. However, the role of mTORC1/2
inhibition in apoptosis induction has not been studied in depth. Whether mTORC1 or mTORC2 inhibition induces the activation of specific pro-apoptotic proteins or causes a metabolic imbalance that primes cells towards apoptosis in the cell is still unknown. Our findings show that low concentrations of AZD8055 and MNL0128 are not highly effective in mice, suggesting that mTORC1/2 inhibition results in a pro-apoptotic state in TC cells. The pro-apoptotic state induces vulnerability to apoptosis induction by chemotherapeutic drugs like cisplatin, making these inhibitors of interest for tumors that have developed resistance to certain cytotoxic drugs. In addition, our results suggest that autophagy plays a protective role against apoptosis in TC cells, indicating that inhibition of mTORC1/2 in combination with autophagy blockade could further enhance sensitivity towards cisplatin in TC tumors. Hydroxychloroquine is the only autophagy inhibitor in clinical use so far. It has been proven to have a safe profile and has been combined with cytotoxic drugs in cancer clinical trials with good tolerability but not in TC patients40.
Patient-derived xenograft models of TC
TC is a rare disease; hence obtaining patient material for implantation in mice was a limitation for our study (Chapter 4). In total, we established three PDX models in immunodeficient mice. To our knowledge this was the first report of TC PDX models developed in NOD SCID gamma (NSG) mice, while other studies have used nude mice and orthotopic implantation. It has been hypothesized that orthotopic implantation is necessary for TC PDX models to establish, however we found similar take rates using subcutaneous implantation (35% vs 27%)29. The advantages of subcutaneous implantation are the obvious
less complicated surgical procedure and the fact that monitoring tumor growth does not require imaging techniques41. To further improve take rates of TC PDX
models, it will be necessary to compare different techniques and conditions that have shown to have an impact on takes rates of other PDX models such as growth factors in transport media or use of matrigel during implantation42, 43.
Moreover, testicular microenvironment consists of different cell types, among those are spermatogonias, Leydig and Sertolli cells. Leydig and Sertolli cells are responsible for secreting hormones like estradiol, testosterone and other paracrine factors that are involved in spermatogenesis and other physiological
processes. Abnormalities in the testis microenvironment have been described in TC patients, more specifically, Leydig cell dysfunction. Whether these abnormalities influence PDX tumor evolution and treatment response is at this point an open question44.
Interestingly, we showed a similar sensitivity towards the drug in TC PDX models as the patient’s response to cisplatin-based chemotherapy (Chapter 4). Our results are in line with previous data showing PDX models have similar sensitivity towards treatment as cancer patients45, 46. Thus, TC PDX models could
be great tools to test treatment strategies before going into clinical trials, as we demonstrated in Chapter 3. An important phenomenon that occurs during PDX model establishment and that has brought much attention is genetic evolution across passages. Our results suggest that TC tumors go through some degree of adaptation while maintaining important cancer features like histological subtypes, heterogeneity and drug response in mice. Further examination of the mutational landscape and evolution through passages is currently being performed on the available TC PDX specimens to gain more insight in features like genetic heterogeneity, tumor adaptation and mutational analysis. Such in depth evaluation of TC PDX tumors could provide novel targets to treat TC more effectively.
Within the poor prognosis group, patients bearing extragonadal nonseminomatous primary tumors have a worse survival17, 19. It has been reported that between 5.5%
and16.3% of all germ cell tumors are extragonadal47. Incidence of these tumors
has not increased over the past decades while incidence of TC has, pointing at a different etiology of the disease and therefore the need of developing and studying extragonadal PDX tumors to gain new insights into their resistance mechanisms and offer new treatment options.
Conclusion
Even though TC is highly curable with cisplatin-based chemotherapy, there are some challenges hampering patient survival that involve patient stratification and resistance to therapy. This thesis provides new insight on the utility of novel biomarkers, specifically miRNAs, to identify TC patients with a higher risk of having a poor outcome. Additionally, novel in vivo TC models were characterized in depth and were used to test a cisplatin-based combination treatment, such as mTORC1/2 inhibitors that were identified in our in vitro experiments. Based on these results, adding mTORC1/2 inhibitors to BEP-chemotherapy in patients with relapse or refractory disease may improve clinical outcome of TC. Early detection of those patients using miRNAs in blood could then assist clinicians in taking additional measures to prevent evolution of TC to a more aggressive disease.
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