2.1 Patients and specimens
The patients included in our study were retrieved from the database of pathological
diagnosis in the Department of Pathology, National Taiwan University Hospital
(NTUH). For cases of low-grade noninvasive papillary urothelial neoplasm, we
recruited all 66 patients diagnosed as PUNLMP of the urinary bladder from 2005 to
2014 and collected their tissue specimens of initial diagnosis. Four patients with the initial diagnosis of “transitional cell carcinoma, grade I” from 2000 to 2004 were also
added to our study cohort. In addition, we randomly selected 26 patients with inverted
papilloma from 2005 to 2014 for comparison. Hematoxylin and eosin (H&E)-stained
sections of all cases were reviewed and reclassified according to the WHO 2016 tumour
classification (Figure 1). Cases without adequate tumor contents for DNA extraction
were excluded from the analysis. After confirming the histologic diagnosis of each case,
the patients’ age, sex, and follow-up data (status and periods of tumor recurrence and
progression) were recorded. Tumor progression was defined as recurrence as high-grade
NIPUC, urothelial carcinoma in situ, or invasive urothelial carcinoma. One
representative section with an adequate tumor part and the corresponding formalin-fixed
paraffin-embedded (FFPE) tissue block were selected for each case. This part of study
th
201508043RIND; revised on Dec 30th, 2016).
For MIBC cases, we included all 109 patients who underwent radical cystectomy
from 2010 to 2016 and collected their clinical data. Patients with a history of UTUC
were excluded to avoid confounding prognostic analysis. In each patient who received
neoadjuvant chemotherapy, the latest resection specimen prior to cystectomy was
selected for this study. In this group, those who did not have a preoperative specimen
available at NTUH were excluded from this study. In patients without neoadjuvant
chemotherapy, the cystectomy specimens were selected. Alternatively, the latest
resection specimen before cystectomy was selected if no adequate tumor cells were
present in the cystectomy specimen. H&E-stained sections of all cases were reviewed
and re-staged by the TNM classification system defined in the AJCC Cancer Staging
Manual (8th edition). Cases without definite muscularis propria invasion were excluded
at this point. One representative section with an adequate tumor part and the
corresponding FFPE tissue block were selected for each case. This part of study was
approved by the Research Ethics Committee of NTUH on Oct 16th, 2017 (No.
201708055RIND; revised on Aug 17th, 2018).
2.2 DNA extraction and sequencing
For each case with low-grade noninvasive papillary urothelial neoplasm, five 10-μm
unstained slides were sectioned from the representative tissue block. DNA was extracted
using the QiaAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) following the
manufacturer’s instructions. The mutation hotspots in the promoter region of the TERT
gene, 3 exons of coding regions in the FGFR3 gene, and 1 exon in the HRAS gene were
amplified through polymerase chain reaction (PCR). The amplicons of the FGFR3 gene
included codons 248 and 249 in exon 7, codons 372 and 375 in exon 10, and codon 652
in exon 15. A similar assay in the HRAS gene focused on codon 61 in exon 3. We
selected these exons of the target genes based on previous reports showing the frequent
mutated genes in urothelial neoplasms and their mutational hot spots [6–19]. The
primers for PCR are listed in Table 1, and the Sanger method was used to sequence the
PCR products.
2.3 IHC staining
For each case with MIBC, 5-μm sections were taken from the representative tissue
block for IHC staining. The staining procedures were conducted with a Ventana
Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to
the manufacturer’s instructions. Primary antibodies against p53 (clone DO-7, dilution
1:1000, Dako Denmark A/S, Glostrup, Denmark), GATA3 (clone L50-823), CK20
(clone SP33), CK5/6 (clone D5/16B4), and Ki-67 (clone 30-9) were included. All
antibodies other than anti-p53 antibody were purchased from Ventana Medical Systems
and were ready to use. The antibody reactivity was visualized with a Ventana OptiView
DAB IHC Detection Kit. Finally, the slides were counterstained with hematoxylin.
All IHC results were examined under a light microscope and scored under the
following criteria. The percentage and intensity of tumor cells stained with GATA3
(nuclear staining), CK20, and CK5/6 (membranous-type or cytoplasmic staining) were
recorded for each case and their immunoreactive scores (IRS) were calculated using
Remmele and Stegner’s criteria [47, 48] (Table 2). According to the cut-offs used in
IRS, their staining percentage was also classified as negative (<10%), partial
(10%–80%), and diffuse (>80%). The standards for p53 scores are demonstrated in
Figure 2 by combining the criteria used in prior studies of bladder [44] and ovarian [45,
49] cancers. The Ki-67 indices were evaluated by following the recommendations from
the International Ki67 in Breast Cancer Working Group [50]. In brief, at least three
400× fields containing 500 or more invasive tumor cells were selected for each case.
Tumor cells with nuclear staining were considered positive regardless of the staining
intensity. The Ki-67 index was calculated as the percentage of the positive cells among
the total number of tumor cells in the scored area.
2.4 Clinicopathological correlation and survival analysis
Student t test and one-way analysis of variance (ANOVA) were used to evaluate
differences in the continuous parameters between or among comparable groups.
Chi-square test and Spearman rank correlation test were applied to analyze the
association among categorical and continuous parameters, respectively. For cases of
PUNLMP and low-grade NIPUC, we calculated the cumulative recurrence-free survival
(RFS) and PFS from the point of initial diagnosis by using the Kaplan–Meier method.
In chemotherapy-naïve MIBC patients, we calculated OS and DSS after radical
cystectomy in addition to RFS. The differences in survival time were determined using
log-rank tests. For MIBC patients, Cox regression was used to determine the association
between continuous parameters and clinical outcomes. Multivaraite analyses were also
performed using Cox regression. P < 0.05 was considered statistically significant. Cox
regression was performed with SAS (version 9.4; SAS Institute Inc., Cary, NC, USA)
with the assistance of the Department of Medical Research, NTUH, and the other
statistical analyses were conducted using Microsoft Excel 2007 and Prism (version 7.03;
GraphPad Software, Inc., La Jolla, CA, USA).