A case-control exploratory study of testicular cancer survivors (TCS) treated with chemotherapy matched by age and gender with healthy controls. Cases were defined as TCS, 18 years old or older, who were in surveillance and no evidence of disease (NED) ≥ 3 months (negative tumor markers and computed tomography (CT) image with no evidence of disease after the last oncologic treatment), and who had received at least 3 BEP (bleomycin, etoposide, and cisplatin) cycles. Patients treated with high dose chemotherapy and bone marrow transplant were excluded. Controls were healthy males, with no previous history of cancer, matched by age (+/- 12 months) in a 1:1 ratio. Before the inclusion in the study and in order to be considered “healthy”, all participants were evaluated clinically by an Internal Medicine specialist (MTB) and screened for type 2 diabetes mellitus and dyslipidemia.
This study was approved by the Institutional Biomedical Research Board of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (REF. 1785). All subjects were informed about the objectives of the study and gave their written consent to participate.
Isolation of peripheral blood mononuclear cells (PBMC)
A sample of venous blood (40 mL) was obtained from each subject. PBMC were isolated by gradient centrifugation using Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). Researchers were blinded to the origin of the sample cases vs controls.
CD3-mAb-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to purify CD3+ cells by positive selection following the manufacturer’s instructions. Purity was assessed by flow cytometry with an anti-human CD3-FITC monoclonal antibody. This procedure normally yielded CD3+ T-cell preparations with purity >95%.
RNA extraction and cDNA synthesis
Total RNA from CD3+ lymphocytes was obtained using Trizol (Life Technology, New York, USA) according to the manufacturer's instructions. cDNA was synthesized from total RNA by using random hexamers as primers and murine leukemia virus reverse transcriptase (RT) following the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA).
Expression of CDKN2A/p16INK4a
The expression of CDKN2A/p16INK4a was measured using the qPCR Taqman assay (TaqMan Universal Master Mix II, with UNG, Applied Biosystems, Foster City, USA) according to the manufacturer's specifications. TaqMan probes were used for CDKN2A/p16INK4a (P16-FAM HS_00924091), Applied Biosystems, Foster City, USA) and 18S (18S-VIC HS_99999901) (Applied Biosystem, Foster City, USA). The samples were performed in duplicate in the real-time polymerase chain reaction (RT-PCR) equipment Corbett Research model RG-6000 (Sydney, Australia) using the program Roto-gene 6000 version 1.7. The relative expression in cases vs controls of CDKN2A/p16INK4a was analyzed using the 2-ΔΔCt method comparing each case with a matched control.
Immunophenotyping of leukocyte subpopulations
EDTA-treated blood samples were analyzed by 8-color flow-cytometry (Becton Dickinson Canto II Cytometer) using fluorescence-labelled antibodies from Biolegend Inc. (San Diego, USA). Briefly, 250 µL of blood was incubated with fluorochrome-conjugated antibodies for 20 min at room temperature prior to lysis (RBC Lysis Buffer, Biolegend Inc., San Diego, USA) and fixed with 3% formaldehyde/PBS. Leukocytes populations were defined by the following marker combinations: B cells CD3- CD19+, T cells CD3+, CD4 T cells CD3+CD4+, CD8 T cells CD3+CD8+, CD4 naïve CD4+CD45RA+CD197+, CD4 central memory (TCM) CD4+CD45RO+CD197+, CD4 effector memory (TEM) CD4+CD45RO+CD197-, CD8 naïve CD3+CD8+CD45RA+CD197+, CD8 TCM CD3+CD8+CD45RO+CD197+, CD8 TEM CD3+CD8+CD45RO+CD2197-, NKT cells CD4+CD56+CD16high, NK cells CD4-CD56+, naïve B cells CD19+CD20+CD27-, Memory B cells CD19+CD20+CD27+, Plasmablasts CD19+CD20-CD27+CD38high, CD4 Treg cells CD4+CD127lowCD25+, and CD8 Treg cells CD8+CD28-.
OneFlow™Setup Beads (BD Biosciences, San Jose, CA, USA) were used to adjust instrument settings, set fluorescence compensation, and check instrument sensitivity. ‘Fluorescence minus one’ controls were used to determine positive and negative staining boundaries for each fluorochrome. Five hundred thousand events were recorded for each sample and analyzed with the FlowJo® software v.10. (FlowJo, LLC., Ashland, OR). For initial gating, singlets were identified using the FSC-Height (FSC-H) by FSC-Area (FSC-A) scatter plot. Then the lymphocyte population was gated in a plot SSC-A versus FSC-A. From there, subsequent gating was designed to identify major populations.
Cases and controls were compared with the Wilcoxon signed-rank test, a p-value ≤0.05 was considered statistically significant. Results are expressed as median and interquartile range (IQR) unless otherwise indicated. For analysis of significance of the relative expression of CDKN2A/p16INK4a in cases vs controls, Wilcoxon signed rank test was used. SPSS® version 21.0 and GraphPad prism v.7.05 were used for the analysis.