Are Changes in the Percentage of Specific Leukocyte Subpopulations Associated with Endogenous DNA Damage Levels in Testicular Cancer Patients?

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.

Increased levels of XPA might be the basis of cisplatin resistance in germ cell tumours.

BACKGROUND: Germ cell tumours (GCTs) represent a highly curable malignity as they respond well to cisplatin (CDDP)-based chemotherapy. Nevertheless, a small proportion of GCT patients relapse or do not respond to therapy. As this might be caused by an increased capacity to repair CDDP-induced DNA damage, identification of DNA repair biomarkers predicting inadequate or aberrant response to CDDP, and thus poor prognosis for GCT patients, poses a challenge. The objective of this study is to examine the expression levels of the key nucleotide excision repair (NER) factors, XPA, ERCC1 and XPF, in GCT patients and cell lines. METHODS: Two hundred seven GCT patients' specimens with sufficient follow-up clinical-pathological data and pairwise combinations of CDDP-resistant and -sensitive GCT cell lines were included. Immunohistochemistry was used to detect the ERCC1, XPF and XPA protein expression levels in GCT patients' specimen and Western blot and qRT-PCR examined the protein and mRNA expression levels in GCT cell lines. RESULTS: GCT patients with low XPA expression had significantly better overall survival than patients with high expression (hazard ratio = 0.38, 95% confidence interval: 0.12-1.23, p = 0.0228). In addition, XPA expression was increased in the non-seminomatous histological subtype, IGCCCG poor prognosis group, increasing S stage, as well as the presence of lung, liver and non-pulmonary visceral metastases. Importantly, a correlation between inadequate or aberrant CDDP response and XPA expression found in GCT patients was also seen in GCT cell lines. CONCLUSIONS: XPA expression is an additional independent prognostic biomarker for stratifying GCT patients, allowing for improvements in decision-making on treatment for those at high risk of refractoriness or relapse. In addition, it could represent a novel therapeutic target in GCTs.

DNA damage levels in peripheral blood mononuclear cells before and after first cycle of chemotherapy have comparable prognostic values in germ cell tumor patients.

Background: Germ cell tumors (GCTs) represent the most frequent solid malignancy in young men. This malignancy is highly curable by cisplatin (CDDP)-based chemotherapy. However, there is a proportion of patients having a poor prognosis due to refractory disease or its relapse. No reliable biomarkers being able to timely and accurately stratify poor prognosis GCT patients are currently available. Previously, we have shown that chemotherapy-naïve GCT patients with higher DNA damage levels in peripheral blood mononuclear cells (PBMCs) have significantly worse prognosis compared to patients with lower DNA damage levels. Methods: DNA damage levels in PBMCs of both chemotherapy-naïve and first cycle chemotherapy-treated GCT patients have been assessed by standard alkaline comet assay and its styrene oxide (SO)-modified version. These levels were correlated with clinico-pathological characteristics. Results: We re-confirm prognostic value of DNA damage level in chemotherapy-naïve GCT patients and reveal that this prognosticator is equally effective in GCT patients after first cycle of CDDP-based chemotherapy. Furthermore, we demonstrate that SO-modified comet assay is comparably sensitive as standard alkaline comet assay in case of patients who underwent first cycle of CDDP-based chemotherapy, although it appears more suitable to detect DNA cross-links. Conclusion: We propose that DNA damage levels in PBMCs before and after first cycle of CCDP-based chemotherapy are comparable independent prognosticators for progression-free and overall survivals in GCT patients. Therefore, their clinical use is highly advised to stratify GCT patients to identify those who are most at risk of developing disease recurrence or relapse, allowing tailoring therapeutic interventions to poor prognosis individuals, and optimizing their care management and treatment regimen.

Differential gene expression in cisplatin-resistant and -sensitive testicular germ cell tumor cell lines.

Testicular germ cell tumors (TGCTs) represent a well curable malignity due to their exceptional response to cisplatin (CDDP). Despite remarkable treatment results, approximately 5% of TGCT patients develop CDDP resistance and die. Exceptional curability makes TGCTs a highly valuable model system for studying the molecular mechanisms of CDDP sensitivity. Our study was aimed at revealing difference in gene expression between the CDDP-resistant and -sensitive TGCT cell lines, and hence at identifying candidate genes that could serve as potential biomarkers of CDDP response. Using gene expression array, we identified 281 genes that are differentially expressed in CDDP-resistant compared to -sensitive TGCT cell lines. The expression of 25 genes with the highest fold change was validated by RT-qPCR. Of them, DNMT3L, GAL, IGFBP2, IGFBP7, L1TD1, NANOG, NTF3, POU5F1, SOX2, WNT6, ZFP42, ID2, PCP4, SLC40A1 and TRIB3, displayed comparable expression change in gene expression array and RT-qPCR, when all CDDP-resistant TGCT cell lines were pairwise combined with all -sensitive ones. Products of the identified genes are pluripotency factors, or are involved in processes, such as cell metabolism, proliferation or migration. We propose that, after clinical validation, these genes could serve as prognostic biomarkers for early detection of CDDP response in TGCT patients.

DNA damage measured in blood cells predicts overall and progression-free survival in germ cell tumour patients.

Germ cell tumour (GCT) patients who fail to respond to chemotherapy or who relapse have a poor prognosis. Timely and accurately stratifying such patients could optimise their therapy. We identified endogenous DNA damage levels as a prognostic marker for progression-free (PFS) and overall (OS) survival in chemotherapy-naïve GCT patients. In the present study, we have extended our previous results and reviewed the prognostic power of DNA damage level in GCTs. Endogenous DNA damage levels were measured with the comet assay. Receiver operator characteristic analysis was applied to determine the optimal cut-off value and to evaluate its prognostic accuracy. PFS and OS were estimated by the Kaplan-Meier method and compared using the log-rank test. Hazard ratio (HR) estimates were calculated by Cox regression analysis. A cut-off value of 6.34 provided the highest sensitivity and specificity, with area under curve values of 0.813 and 0.814 for disease progression and mortality, respectively. A % DNA in tail > 6.34 was significantly associated with shorter PFS (HR = 9.54, 95 % confidence interval [CI]: 3.43-26.55, p < 0.001) and OS (HR = 14.62, 95 % CI: 3.14-67.95, p = 0.001) by univariate analysis. The prognostic value of DNA damage measurement was confirmed by multivariate models (HR = 6.45, 95 % CI: 2.22-18.75, p = 0.001 for PFS and HR = 9.40, 95 % CI: 1.70-52.09, p = 0.010 for OS), when HR was adjusted for relevant clinical categories. The added prognostic value of DNA damage in combination with International Germ Cell Cancer Collaborative Group (IGCCCG) risk groups has been revealed. Endogenous DNA damage is an independent prognosticator for PFS and OS in GCT patients and its clinical use, particularly in combination with IGCCCG risk groups, may help in stratifying these patients.

High Endogenous DNA Damage Levels Predict Hematological Toxicity in Testicular Germ Cell Tumor Patients Treated With First-Line Chemotherapy.

BACKGROUND: Testicular germ cell tumors (TGCTs) are an excellent example of chemosensitive disease. However, cisplatin-based chemotherapy has significant side effects, including myelosuppression. Previously, we found endogenous DNA damage level in peripheral blood mononuclear cells (PBMCs) to be an independent prognostic marker. In this study, we tested the hypothesis that patients with high endogenous DNA damage levels in PBMCs have an increased risk of developing hematological toxicity. PATIENTS AND METHODS: One hundred twenty chemotherapy-naive TGCT patients treated in the National Cancer Institute and the St Elisabeth Cancer Institute in Bratislava, Slovakia, from 2012 to 2018 were enrolled. All patients received platinum-based chemotherapy with granulocyte colony stimulating factor support. On the day of starting treatment, we measured the DNA damage levels in PBMCs using the comet assay. We used the cutoff level of 5.25, a value previously reported to stratify patients on the basis of their prognosis. We monitored hematological toxicity during the first cycle of chemotherapy. The mean and standard error of the mean were calculated for all variables. RESULTS: Patients with high DNA damage levels (>5.25) had more significant hematological toxicity with significantly lower nadir white blood cell count (P = .001), absolute neutrophil count (P = .013) and absolute lymphocyte count (ALC; P < .001). ALCs on day 0 (P = .005) and day 22 (P = .046) were also significantly lower in patients with high DNA damage levels. CONCLUSION: This study shows that higher endogenous DNA damage levels correlate with increased risk of hematological toxicity in TGCT patients. Hence, the DNA damage levels can be used to select patients for closer monitoring because of a higher risk of acute chemotherapy-related complications.

The prognostic value of DNA damage level in peripheral blood lymphocytes of chemotherapy-naïve patients with germ cell cancer.

Germ cell tumors (GCTs) are extraordinarily sensitive to cisplatin (CDDP)-based chemotherapy. DNA damage represents one of the most important factors contributing to toxic effects of CDDP-based chemotherapy. This study was aimed to evaluate the prognostic value of DNA damage level in peripheral blood lymphocytes (PBLs) from chemo-naïve GCT patients. PBLs isolated from 59 chemotherapy-naïve GCT patients were included into this prospective study. DNA damage levels in PBLs were evaluated by the Comet assay and scored as percentage tail DNA by the Metafer-MetaCyte analyzing software. The mean ± SEM (standard error of the mean) of endogenous DNA damage level was 5.25 ± 0.64. Patients with DNA damage levels lower than mean had significantly better progression free survival (hazard ratio [HR] = 0.19, 95% CI (0.04 - 0.96), P = 0.01) and overall survival (HR = 0.00, 95% CI (0.00 - 0.0), P < 0.001) compared to patients with DNA damage levels higher than mean. Moreover, there was significant correlation between the DNA damage level and presence of mediastinal lymph nodes metastases, IGCCCG (International Germ Cell Cancer Collaborative Group) risk group, and serum tumor markers level. These data suggest that DNA damage levels in PBLs of GCT patients may serve as an important prognostic marker early identifying patients with poor outcome.