3.1 Mutation status in each histological entity of low-grade noninvasive papillary
urothelial neoplasms
After review of all 96 cases with low-grade noninvasive papillary urothelial neoplasms,
4 were reclassified as high-grade NIPUC and excluded from our study. Four additional
cases were excluded because the tissue specimens of initial diagnosis were not available
or limited in amount. In the mutation analyses, PCR for TERT promoter sequences were
unsuccessful in 3 cases after two repeated experiments. These 3 cases were also
excluded. The 85 cases finally included in this study comprised 21 inverted papillomas,
30 PUNLMPs, and 34 low-grade NIPUCs. The clinical characteristics of patients and
the results of the mutation status in each entity were summarized in Table 3.
TERT promoter mutations were found in 33% (10 of 30) of PUNLMP, 50% (17 of
34) of low-grade NIPUC and none of the 21 inverted papillomas. The rates of FGFR3
mutations were also much higher in PUNLMP (30%, 9 of 30) or low-grade NIPUC
(50%, 17 of 34) than in inverted papilloma (10%, 2 of 21) (p = .009). Conversely, HRAS
mutations were more frequently observed in inverted papilloma (76%, 16 of 21) than in
PUNLMP (10%, 3 of 30) or low-grade NIPUC (15%, 5 of 34) (p < .001). We compared
the mutation frequency between PUNLMP and low-grade NIPUC groups and found no
Regarding mutation patterns, the most frequent point mutation in the TERT
promoter region was C228T (19 of all 27 mutated cases), followed by C250T (6 of 27
cases) and C228A (2 of 27 cases) (Figure 3, upper panel). The most common mutation
in FGFR3 was S249C (16 of all 28 mutated cases), followed by Y375C (7 of 28 cases)
(Figure 3, middle panel). Other mutations in FGFR3 included G372C (1 case), S373C
(1 case), K652E (1 case), and K652T (2 cases). Mutations in the HRAS gene included
Q61R (14 of all 24 mutated cases), Q61K (7 cases), and Q61L (3 cases) (Figure 3,
lower panel) (Table 4).
3.2 Prognostic significance of the mutation status in PUNLMP and low-grade
NIPUC
The PUNLMP and low-grade NIPUC patients were followed at our outpatient clinics
with a median follow-up period of 5.7 years, and most (61 of 64, 95.3%) cases have
been followed for more than 2 years. We first analyzed the clinical outcomes of all
patients with PUNLMP or low-grade NIPUC. Patients with low-grade NIPUC had
shorter RFS than those with PUNLMP (p = 0.002, Figure 4A), and the TERT promoter
mutation status had borderline significance regarding RFS in these patients (p = 0.052,
Figure 4B). We then separately analyzed the survival data of 30 patients with PUNLMP
and 34 patients with low-grade NIPUC. In the PUNLMP group, patients with TERT
promoter mutations had shorter RFS than those without mutations (p = 0.024, Figure
4C). In contrast, the TERT promoter mutation status was not related to RFS in the
low-grade NIPUC group (p = 0.530, Figure 4D).
Tumor progressions occurred in only three patients. One patient progressed from
PUNLMP to high-grade NIPUC and then high-grade invasive urothelial carcinoma. The
other patient initially diagnosed as PUNLMP progressed to high-grade invasive
urothelial carcinoma directly. The third patient progressed from low-grade NIPUC to
high-grade NIPUC and then high-grade invasive urothelial carcinoma. Neither the
histologic entity nor the TERT promoter mutation status had significant impact on PFS.
In addition, mutation status of the FGFR3 gene has no prognostic association in
PUNLMP, low-grade NIPUC, or combined. Analysis of the association between
survival and the mutation status of HRAS was not performed due to the limited number
of mutated cases in each group.
3.3 Prognostic grouping by combination of histological classification and mutation
status of TERT promoter
Because of the prognostic significance of the TERT promoter mutation in PUNLMP, we
further categorized PUNLMP and low-grade NIPUC cases into 4 groups based on the
mutation status of the TERT promoter. Group 1 was defined as PUNLMP cases without
TERT promoter mutations, and group 2 represented those with the mutations.
Low-grade NIPUC cases with wild-type and mutated TERT promoter regions were
referred to as groups 3 and 4 respectively. We then compared the Kaplan-Meier curves
of RFS among these groups, and the result was shown in Figure 4E. The recurrence rate
of PUNLMP without TERT promoter mutations (group 1) was significantly lower than
other groups (overall p = 0.007). Moreover, there was no significant difference in RFS
among PUNLMP with TERT promoter mutations (group 2) and low-grade NIPUC cases
(groups 3 and 4; p = 0.487).
3.4 Demographic and clinicopathological data regarding patients with MIBC
Among the 109 patients recruited for our MIBC study, definite muscularis propria
invasion (i.e. stage pT2 or higher) was not identified in 9, the microscopic slides were
unavailable in 7, a history of UTUC was noted in 1, and loss to follow-up after radical
cystectomy was noted in 1; these 18 cases were all excluded. Finally, we included 91
patients (median age: 67 years [range: 39–89 years]; male-to-female ratio: 2.37:1). Of
them, 13 (14.3%) received neoadjuvant chemotherapy. In patients not receiving
neoadjuvant chemotherapy, the median follow-up time was 2.46 years. The
demographic and clinicopathological data of these patients are summarized in Table 5.
3.5 Association among GATA3, CK20 and CK5/6 staining in MIBC
After completion of IHC, we evaluated the correlation of the staining results among
each marker. The results are summarized in Table 6. First, CK20 and GATA3
demonstrated a positive correlation in terms of both percentage and IRS score. The
percentage of GATA3 staining tended to be higher than that of CK20, and CK20
staining was stained on GATA3-positive tumor cells in most cases. By contrast, a
negative correlation was observed between CK20 and CK5/6. In cases with both CK20
and CK5/6 reactivity, the staining patterns of these two markers were generally
reciprocal. Although they were not completely mutually-exclusive, CK20 tended to be
stained on CK5/6-negative tumor cells and vice versa. Figure 5A illustrates two
examples of such a reciprocal pattern. Of the 21 (23.08%) tumors with minimal
(1%–9%) CK5/6 staining, 5 (5.49%) demonstrated basal alignment in the aggregates of
tumor cells. Because the case number was limited, analyzing the biological significance
of such a pattern is unfeasible.
Similar to CK20, GATA3 showed negative correlation with CK5/6 expression.
However, coexpression of GATA3 and CK5/6 was observed in 44 (48.35%) cases; of
them 13 (14.29%) had diffuse coexpression (staining in >80% of tumor cells for both
markers). Two examples of GATA3/CK5/6 coexpression are illustrated in Figure 5B. In
the 13 double-positive cases, 11 (84.62%) showed completely absent or minimal
staining for CK20. Meanwhile, double-negative tumors for GATA3 and CK5/6
accounted for 3 (3.30%) of the 91 cases, and only one of them showed complete
absence for each marker.
3.6 Association of Ki-67 index with GATA3, CK20, CK5/6 and p53 expression in
MIBC
As presented in Table 6, Ki-67 indices were significantly correlated with staining
percentages of GATA3, CK20, and CK5/6. We further categorized each of the latter
three markers into three groups (negative, partial, and diffuse) and compared their
difference through Ki-67 indices. Differences in Ki-67 indices was also compared
among each group of the p53 score. The results are summarized in Table 7. In brief,
tumors with diffuse GATA3 staining had significantly lower Ki-67 indices. By contrast,
high Ki-67 indices were observed in negative CK20 and diffuse CK5/6 cases. The
difference between negative and partial staining groups of GATA3 (p = 0.616) or CK5/6
(p = 0.565) was nonsignificant. Similarly, no difference was observed between cases
with partial and diffuse CK20 reactivity (p = 0.986).
The association between p53 score and the Ki-67 index was more complex.
Absence of p53 staining (score 0) was associated with a significantly higher Ki-67
index compared with the partial staining group (score 1, p = 0.004). In addition, the
score 2 group had a significantly lower Ki-67 index than the score 3 group (p < 0.001),
but no difference was found between the score 1 and 2 groups (p = 0.079). In addition,
no difference was shown between the p53-absent (score 0) and score 3 groups (p =
0.164). These findings were consistent with the definition of aberrant p53 expression in
ovarian carcinoma [41]. Notably, 1 (1.1%) case in our cohort showed diffuse
cytoplasmic staining with variable nuclear intensity (score 4). This pattern was found to
be associated with TP53 mutation in a previous study on ovarian carcinoma [49] and
considered p53-aberrant. Based on this definition, tumors with aberrant p53 staining had
significantly higher Ki-67 indices (p< 0.001). No difference in GATA3, CK20, or
CK5/6 expression was noted between p53-aberrant and non-p53-aberrant tumors.
3.7 Intratumoral heterogeneity in MIBC
In case RC01-22, a minor component (2% of total tumor area) with significantly
different morphology was observed in the tumor. Although the major part showed
considerable squamous differentiation, this minor component had usual histology of UC
with heavy lymphocytic infiltration. As for the IHC markers, the major part expressed a
typical basal/squamous profile of diffuse CK5/6 staining, minimal GATA3 reactivity,
and a non-aberrant pattern of p53. By contrast, the minor component was partially
positive for both GATA3 and CK5/6 with diffusely strong reactivity to p53 (Figure 6).
This patient demonstrated tumor recurrence after radical cystectomy, but the diagnosis
of recurrence was based on radiologic images without acquisition of tissue specimens.
For analytical purposes, the IHC profile of the major part was used for this case. No
other tumor with apparent intratumoral heterogeneity was noted in our MIBC study
cohort.
3.8 Prognostic significance of the IHC markers in MIBC
Among the IHC markers (GATA3, CK20, CK5/6, p53 and Ki-67), only GATA3
demonstrated significant correlation with clinical outcomes. In the 78 patients without
neoadjuvant chemotherapy, higher percentage of GATA3 staining was associated with a
significantly better RFS in both univariate (p = 0.008) and multivariate (p = 0.002)
analysis by using Cox regression (Table 8). Analysis by IRS revealed similar results
(Table 9). In the Kaplan-Meier plot, gradual difference in RFS was present among cases
with diffuse, partial, and negative GATA3 staining (p = 0.002). The other significant
prognostic parameters included the T stage (for RFS) and the presence of nodal
metastasis (for DSS and RFS). The Kaplan–Meier plots for GATA3, T stage, and nodal
metastasis are depicted in Figure 7.