Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) as a novel marker in T1 high-grade and T2 bladder cancer patients receiving neoadjuvant chemotherapy

The cohort under investigation comprised 44 patients who underwent neoadjuvant chemotherapy for pT1 high-grade or pT2N0M0 bladder cancer at our institution between April 2003 and February 2011. Having been compiled for research purposes, this group represents patients for whom pretreatment, archival paraffin-embedded tissue blocks and data from complete clinical follow-up were available. Diagnostic work-up included initial transurethral resection of bladder tumor (TURBT), pelvic magnetic resonance imaging (MRI), chest and abdominal computed tomography (CT), and bone scintigraphy. Tumors were graded histologically in accordance with World Health Organization (WHO) classifications and were staged as per the TNM staging system of the Union for International Cancer Control (2009). Histological type was urothelial carcinoma in all cases.

Neoadjuvant intra-arterial chemotherapy was performed after complete TURBT, only after the patient consented to therapy based on our recommendation. Written informed consent was obtained from all patients. Anticancer agents administered as neoadjuvant chemotherapy consisted of cisplatin at 100 mg/m², methotrexate at 30 mg/m², and doxorubicin at 20 mg/m². Our therapeutic protocol comprised two courses of neoadjuvant chemotherapy. Following this, a second TURBT was performed to obtain a biopsy specimen. We assessed the efficacy of neoadjuvant chemotherapy using the pathological results of TURBT. Complete response (CR) was defined as T0 (no evidence of tumor), Ta (noninvasive papillary tumor), or Tis (tumor at a site distant from the original tumor), as in RTOG 99–06 [15]. In cases of CR on the second TURBT, the bladder was preserved, while advanced cases and cases with residual invasive bladder tumors were treated by total cystectomy or systemic chemotherapy [16].

After the second TURBT, cystoscopy and urinary cytological examinations were performed every 3 months for 2 years, every 6 months from 3–5 years, and annually thereafter. Chest radiography and pelvic CT were performed every 6 months for 3 years, and annually thereafter. In cases with visible tumors or hyperemic mucosa in the bladder on cystoscopy or pelvic urinary cytological findings, transurethral biopsy was performed to detect disease recurrence.

This study was carried out in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Approval of the protocol was obtained from the Institutional Review Board of Nippon Medical School, Tokyo, Japan.

DYRK2 expression was determined by immunohistochemical (IHC) staining of paraffin-embedded tissue sections from TURBT specimens immediately before neoadjuvant chemotherapy. The 3-μm-thick sections were deparaffinized, rehydrated using xylene and alcohol, and incubated with 0.3 % H₂O₂ to block endogenous peroxidase activity. Before immunostaining, antigen retrieval was performed at 120 °C for 10 min in an autoclave with citrate buffer (pH 6.0). Staining with a polyclonal anti-DYRK2 antibody (AP7534a; dilution, 1:50; Abgent, San Diego, CA, USA) was performed overnight at 4 °C. Histofine Simple Stain Rabbit MAX PO (MULTI; Nichirei, Tokyo, Japan) was used as the secondary antibody in accordance with the manufacturer’s instructions. Color was developed using diaminobenzidine with 0.01 % H₂O₂. Hematoxylin was used as a counterstain. Stained tumor tissues were evaluated blindly with respect to clinical patient data. Cytoplasmic staining was considered positive, and staining intensity was scored as 0, 1, 2, or 3, corresponding to no staining, weak, moderate, and strong intensities, respectively (Fig. 1). Percentage scores of cells showing cytoplasmic staining were also counted (0–100 %). Total histochemical score (H-score) was calculated by multiplying the intensity score by the percentage score (0–300). An H-score higher than the median was considered positive. Negative controls were incubated without the primary antibody.

Total RNA from formalin-fixed paraffin-embedded tissues was isolated using an Allprep DNA/RNA kit (Qiagen, Tokyo, Japan). The quantity and quality of RNA were evaluated by spectrophotometry. Reverse transcription of RNA to cDNA was achieved using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative gene expression was determined for DYRK2 (Hs00705109_s1) and 18 s (Hs03928990_g1) using gene-specific probes (Applied Biosystems) using TaqMan Fast Advanced Master Mix and the 7900HT Fast Real-time PCR system (Applied Biosystems). PCR conditions were: 5 °C for 2 min and 95 °C for 20 s, followed by 45 cycles at 95 °C for 1 s and 60 °C for 20 s. Data were then quantified using the comparative Ct method for relative gene expression compared with 18S as an endogenous control.

Associations between DYRK2 expression and clinicopathological factors were analyzed using the Fisher’s exact test. Disease-specific survival rates were calculated using the Kaplan–Meier method and differences in survival among groups were compared using log-rank testing. We used Cox proportional hazards regression analysis to assess DYRK2 expression and sex for disease-specific survival. Differences in DYRK2 mRNA between DYRK2-positive and -negative tumors by IHC analysis were determined using the paired t-test. P-values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 21.0 statistical software (IBM Corp, Armonk, NY, USA).