TERT啟動子突變與GATA3、CK20、CK5/6及p53之表現於膀胱泌尿上皮腫瘤之生物學意義

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國立臺灣大學醫學院病理學研究所博士論文

Graduate Institute of Pathology College of Medicine National Taiwan University

Doctoral Dissertation

TERT 啟動子突變與 GATA3、CK20、CK5/6 及 p53 之表現於膀胱泌尿上皮腫瘤之生物學意義

Biological Significance of TERT Promoter Mutation and Expression of GATA3, CK20, CK5/6 and P53 in

Urothelial Neoplasm of the Urinary Bladder

王中傑

Chung-Chieh Wang

指導教授：鄭永銘教授

Advisor: Yung-Ming Jeng, Professor

中華民國 109 年 1 月

January, 2020

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口試委員會審定書

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致謝

猶記八年前剛入學時，我才剛升任主治醫師不久。當時雖然已經習慣於病理診斷工作，對於研究領域卻仍相當陌生。經過張逸良前主任的鼓勵，我報考並就讀了病理學研究所；在鄭永銘教授不厭其煩的指導下，我從對於病理研究完全外行的門外漢，一點一滴地產生了心得與興趣。

這八年來，研究之路其實走得跌跌撞撞。在職場上，出國進修與分院支援讓我一度中斷學業；身體健康的問題，也讓我不得不二度休學。此外，不論是診斷工作還是病理研究，都需要投注相當大量的時間與精力；兩者之間的優先順序安排，常帶給我不小的考驗。我之能夠寫成論文，首先必須感謝鄭永銘教授；除了提供研究的方向與充沛的資源，在計畫撰寫、研究技術與論文寫作方面，鄭教授也給了我全面性的援助與教導。由於論文內容涉及臨床應用，黃昭淵醫師與蔡育傑醫師等臨床先進的指導，對於這篇論文的完成相當重要。此外，感謝賴寶蓮小姐協助切片與蠟塊借調；感謝莊郁琳小姐等研究助理協助進行 DNA 萃取、PCR 與定序工作；感謝切片室與免疫組織化學室同仁在免疫染色的支援；感謝張晉豪博士與統計諮詢團隊在生物統計上的指導。如果沒有各位的努力，這篇論文也不可能完成。

研究的路或許稱不上辛勞，但苦悶與挫折總是在所難免。感謝愛妻柏瑩支持著家庭，讓我能有溫暖而安穩的歸宿；感謝愛女恆音的笑容，在每天的開始帶給我奮鬥的動力。感謝父母的勸勉與鼓勵，幫助我面對各種人生的挑戰；感謝吾弟中瑋與吾友重佑，時時為我分憂解悶。最後，將我的感謝與讚美獻給天父上帝；

願此研究成果能使醫學進步，造福人類，榮耀祢的名。

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中文摘要

膀胱泌尿腫瘤是泌尿科的常見疾病，而其治療主要依據為腫瘤分期與形態學上的分化程度。近期分子病理研究發現了不少重要的腫瘤生物標記，但它們尚未被整合到臨床的治療指引中。本篇論文包含兩部分，分別闡述相關標記於兩類膀胱泌尿腫瘤中的生物學意義：早期的低度非侵襲性泌尿上皮腫瘤，以及晚期的肌肉侵犯性膀胱癌(MIBC)。

在第一部分，我們在低度非侵襲性泌尿上皮腫瘤檢測 TERT、FGFR3 及 HRAS 三種基因的突變狀態，並分析其預後意義。TERT 基因之啟動子突變在膀胱癌頗為 常見，但是在低惡性度乳突狀泌尿上皮腫瘤(PUNLMP)的相關資料並不多。本研究中，我們收集了 21 例良性之倒生性乳突瘤、30 例 PUNLMP，以及 34 例低度非侵 襲性乳突狀泌尿上皮癌(NIPUC)。TERT 基因之啟動子突變出現於 10 (33%)例之 PUNLMP 與 17 (50%)例之低度 NIPUC，但未出現於倒生性乳突瘤。相對於倒生性 乳突瘤，PUNLMP 與低度 NIPUC 較常出現 FGFR3 基因之突變(p = 0.009)，HRAS 基因突變則較為少見(p < 0.001)。在預後方面，PUNLMP 病例有 TERT 啟動子突變 者較容易復發(p = 0.024)，但是低度 NIPUC 並無此現象(p = 0.530)。此外，PUNLMP 病例有 TERT 啟動子突變者，其復發率與低度 NIPUC 無顯著差異(p = 0.487)。

在第二部分，我們分析了 GATA3、CK20、CK5/6 及 p53 在 MIBC 的表現與其生物學意義。近期基因研究將 MIBC 區分為數種子分類，而上述之免疫組織化學染色(IHC)標記與這些子分類有關。其中，GATA3 與 CK5/6 分別被視為管腔型與類 基底-鱗狀型的代表性標記；p53 染色常被用於代替 TP53 基因突變檢測，而傳統上 一般將細胞核染色比例高者視為異常。本研究共收集了 91 名 MIBC 病人的膀胱全切除術組織檢體。在這些病例中，GATA3 表現量較低及 CK20 陰性者，其 Ki-67 增殖指數較高(p 值分別為 0.006 與 0.002)。相對地，CK5/6 呈廣泛性表現者，Ki-67 增殖指數較高(p = 0.001)。P53 染色則有三種類型與高 Ki-67 指數相關：完全陰性、

廣泛的細胞核強染色，以及廣泛的細胞質強染色。此外，CK20 與 CK5/6 的染色結果通常呈現互補關係，但 91 例中的 13 例(14.29%)同時有廣泛的 GATA3 與 CK5/6 表現。在 78 名未接受術前化學治療的病人裡，GATA3 表現量較低者於單變項與多 變項分析均有顯著較高的復發率(p 值分別為 0.008 與 0.002)。CK20、CK5/6 及 p53

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的表現則與預後無關。

總結來說，根據我們的研究結果，TERT 基因的啟動子突變可做為 PUNLMP 病人的預後標記。在 MIBC 病患，GATA3 的表現量則可做為膀胱切除術後的復發危險性指標。此外，由 Ki-67 增殖指數的關聯性來看，以前述三種染色類型做為判斷 p53 染色異常的標準，應比傳統上的核染色比例準則更佳。

關鍵詞：膀胱癌；泌尿上皮腫瘤；TERT 啟動子；GATA3；CK20；CK5/6；p53

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Abstract

Clinical management of bladder urothelial neoplasm depends mainly on the tumor stage

and grade. Recent advances in molecular pathology discovered several essential

biomarkers, and their value in clinical application warrants investigation. In our study,

we focused on the relevant biomarkers in two separate fields of bladder tumors: the

early low-grade noninvasive papillary urothelial neoplasm, and the advanced

muscle-invasive bladder cancer (MIBC).

In the first part, we investigated the mutation status of the TERT promoter, FGFR3

gene, and HRAS gene in low-grade papillary urothelial neoplasms and evaluated their

prognostic significance. Mutations in the promoter region of the TERT gene have been

frequently found in urothelial carcinoma of the urinary bladder, but related data for

papillary urothelial neoplasm of low malignant potential (PUNLMP) are limited. In our

study, we included 21 cases of inverted papillomas, 30 PUNLMPs, and 34 low-grade

noninvasive papillary urothelial carcinomas (NIPUCs). TERT promoter mutations were

observed in 10 (33%) PUNLMPs and 17 (50%) low-grade NIPUCs, but not in any

inverted papilloma. FGFR3 mutations were more frequently observed in PUNLMP and

low-grade NIPUC than in inverted papillomas (p = 0.009), whereas the opposite trend

was noted for HRAS mutations (p < 0.001). Regarding the clinical outcome, TERT

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but not in low-grade NIPUC (p = 0.530). Notably, PUNLMP cases with TERT promoter

mutations had a similar recurrence rate to that in low-grade NIPUC cases (p = 0.487).

Our results suggest that the status of the TERT promoter mutation may serve as a

biomarker of prognostic stratification in patients with PUNLMP.

In the second part, we investigated the biological and prognostic significance of

GATA3, cytokeratin (CK) 20, CK5/6 and p53 in MIBCs from 91 patients who

underwent radical cystectomy. Genetic profiling studies on muscle-invasive bladder

cancers (MIBCs) have discovered several subtypes with different biological

characteristics, and these markers were found to be associated with the molecular

subtypes. According to our results, high Ki-67 indices were associated with negative

CK20 (p = 0.002) and diffuse CK5/6 (p = 0.001) staining. By contrast, tumors with

diffuse GATA3 expression had low Ki-67 index (p = 0.006). Regarding p53, three

staining patterns were associated with a high Ki-67 index: (1) complete absence, (2)

diffusely strong nuclear reactivity, and (3) diffusely strong cytoplasmic staining (p <

0.001 compared with other patterns). CK5/6 and CK20 expression was typically present

in a reciprocal fashion; however, diffuse GATA3 and CK5/6 coexpression was observed

in 13 (14.29%) cases. Among 78 chemotherapy-naïve patients, low GATA3 staining was

associated with worse recurrence-free survival in both univariate (p = 0.008) and

multivariate analyses (p = 0.002). CK20, CK5/6, or p53 expressionwas not associated

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with clinical outcome. Based on our results, IHC staining for GATA3 may help risk

stratification in patients with MIBC receiving radical cystectomy. In addition, the

differences in Ki-67 indices suggested that aberrant p53 expression was better defined

by the three aforementioned patterns, rather than percentage of nuclear staining alone.

Keywords: Bladder cancer; urothelial neoplasm; TERT promoter; GATA3; cytokeratin

20; cytokeratin 5/6; p53

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圖目錄

Figure 1. Representative histological images of inverted papilloma, PUNLMP, and

low-grade NIPUC. ... 44 Figure 2. Scoring criteria for p53 IHC staining ... 45 Figure 3. Common point mutations in the TERT promoter region, FGFR3 gene, and

HRAS gene. ... 46 Figure 4. Kaplan–Meier curves of RFS in patients with low-grade noninvasive papillary

urothelial neoplasms ... 47 Figure 5. (A) Two examples showing reciprocal staining patterns of CK20 and CK5/6

in MIBC. (B) Two examples showing diffuse coexpression of GATA3 and CK5/6 ... 48 Figure 6. Intratumoral heterogeneity in case RC01-22 ... 49 Figure 7. Kaplan–Meier curves regarding the 78 patients with chemotherapy-naïve

MIBC. ... 50

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表目錄

Table 1. Primer sequences for mutation analysis ... 51 Table 2. Immunoreactive score (IRS) by Remmele and Stegner’s criteria ... 52 Table 3. Clinical characteristics and mutation status of patients with inverted papilloma,

PUNLMP, and low-grade NIPUC ... 53 Table 4. Detailed mutation status of inverted papilloma, PUNLMP, and low-grade

NIPUC ... 54 Table 5. Clinicopathological features of the 91 patients who received radical

cystectomy ... 55 Table 6. Correlation among GATA3, CK20, CK5/6, and Ki-67 index ... 56 Table 7. Association between Ki-67 and other IHC markers ... 57 Table 8. Cox regression analysis of clinical outcomes in the 78 chemotherapy-naïve

patients with MIBC using tumor staging and percentages of IHC staining .. 58 Table 9. Cox regression analysis of clinical outcomes in the 78 chemotherapy-naïve

patients with MIBC using tumor staging and immunoreactive scores (IRS) of GATA3, CK20 and CK5/6 staining ... 59

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1. Introduction

1.1 Classification of urothelial neoplasm of the urinary bladder

Urothelial neoplasm of the urinary bladder, or simply bladder tumor, is a major disease

entity commonly encountered by physicians in urology and genitourinary pathology.

This entity encompasses a wide spectrum from the totally benign urothelial papilloma to

advanced bladder cancer. According to the general concept of pathology, the current

version of World Health Organization (WHO) Tumor Classification roughly divides

urothelial neoplasms into the infiltrating urothelial carcinoma and noninvasive

urothelial lesions [1, 2]. The noninvasive neoplasms can be further divided into flat and

papillary lesions by their growth pattern, and the latter includes four categories by the

ascending order of aggressiveness: (1) urothelial papilloma, (2) papillary urothelial

neoplasm of low malignant potential (PUNLMP), (3) low-grade noninvasive papillary

urothelial carcinoma (NIPUC), and (4) high-grade NIPUC [2, 3].

1.2 Clinical management on different categories of the bladder tumors

Clinical management on the urothelial neoplasms depends mainly on the tumor stage

and the histologic grade for noninvasive lesions [4]. From the clinical point of view,

invasion of the bladder tumor into or beyond muscularis propria (stage T2 or more)

warrants aggressive treatment such as radical cystectomy. These advanced tumors are

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also known as muscle-invasive bladder cancers (MIBCs) in the clinical usage and

treatment guidelines [4]. Systemic chemotherapy prior to operation, or neoadjuvant

chemotherapy, may be required in some MIBC patients. By contrast, treatment of

infiltrating urothelial carcinomas limited to the lamina propria (stage T1) is similar to

that of high-grade NIPUC. Although these tumors have potential in progression to

MIBC, most patients require only limited tumor resection, intravesical chemotherapy,

and close clinical follow-up [4].

As their designation implies, patients with low-grade noninvasive papillary lesions

usually follow an indolent clinical course. Urothelial papillomas are totally benign and

do not recur after resection [2]. Low-grade NIPUC has potential of recurrence and even

progression, but most patients require only surgical resection and follow-up [4].

Between them is the entity of PUNLMP, which is characterized by thickening of the

urothelium with minimal cytological atypia [2, 3]. This entity is specific to the urinary

tract and not equivalent to any certain premalignant lesion in other organs. Although it

is associated with a better clinical outcome compared to low-grade NIPUC, it still has

low potential of recurrence. In addition, the differential diagnosis between PUNLMP

and low-grade NIPUC depends only on the degree (minimal versus mild) of nuclear

atypia [2, 3], and this can be subjective in clinical practice. Therefore, WHO Tumor

Classification recommends that patients with PUNLMP should be managed in the same

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manner as those with low-grade NIPUC [2].

1.3 Current problem in management of PUNLMP

Although we already have well-established treatment guidelines for the bladder

urothelial neoplasms, there are still problems to be solved. Current recommendation for

management of PUNLMP is resection and clinical follow-up, but most cases of

PUNLMP never recur or progress (hence the name “low malignant potential”) [2, 3]. In

my speculations, PUNLMP may be further divided into two groups: (1) benign

papillomatous tumors with thickened urothelium, and (2) low-grade NIPUC with

minimal nuclear atypia. Identification of the first group can avoid unnecessary clinical

follow-up in these patients, but these two groups cannot be distinguished by

morphology alone. Genetic studies may provide clues for classification, and knowledge

about the common genetic changes in PUNLMP becomes important in design of our

studies.

1.4 Common gene mutations in low-grade urothelial neoplasms

As the evidence from genetic studies suggests, urothelial neoplasms are likely to

develop through two different routes: high-grade and low-grade pathways [1, 5].

Histologic characteristics of the tumors are often parallel to their genetic changes, as

inactivating mutations of the TP53 gene and subsequent genomic instability are more

commonly seen in tumors with high-grade features [1, 5]. By contrast, mutations

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involving the tyrosine kinase receptor gene FGFR3 and the oncogene HRAS are mainly

associated with the more indolent low-grade papillary urothelial neoplasms [1, 2, 5]. In

addition to these grade-specific alterations, TERT promoter mutations were found in a

considerable percentage of tumor samples regardless of the tumor stage and histologic

grade [6–9]. Details about the three frequently-mutated genes in low-grade urothelial

neoplasms (TERT, FGFR3, and HRAS) are described in the following sections.

1.4.1 TERT promoter mutations

The TERT gene encodes the human telomere reverse transcriptase, which maintains the

telomere length in tumor cells. Mutations in the promoter region of the TERT gene have

been found in not only bladder cancer but also other types of human cancers, including

malignant melanoma, thyroid carcinoma, and glioma [6]. In bladder cancer, C228T was

reported to be the most common mutation pattern in the TERT promoter [7]. In the two

series included by Allory et al., TERT promoter mutations were identified in 70% and

79% of the bladder cancer samples, respectively [7]. Vinagre et al. found TERT

promoter mutations in 67% and 56% of low-grade and high-grade tumors, respectively

[6]. Hosen et al. reported that the rate of the TERT promoter mutation was 66.5% in 158

NIPUC cases, which is similar to a mutation rate of 65.4% in 327 combined cases of all

stages of bladder cancer [8]. Allory et al. also found that TERT promoter mutations

were not significantly associated with the histologic grade, tumor stage, or clinical

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outcome [7].

The data of TERT promoter mutations described above included samples with

different grades and stages, and studies focused on low-grade papillary urothelial

neoplasms are limited. Cheng et al. reported the presence of TERT promoter mutations

in PUNLMP (15 of 35 cases), benign urothelial papilloma (12 of 26 cases) [10], and

inverted papilloma (4 of 26 cases) [9]. Rodriguez Pena et al. collected 30 de novo cases

of PUNLMP and found TERT promoter mutations in 19 (63%) of them. In addition,

there was no association between TERT promoter mutations and the recurrence rate [11].

Taken together, the biological significance of TERT promoter mutations in these

low-grade urothelial lesions remains elusive according to current evidence.

1.4.2 FGFR3 mutations

FGFR3 gene encodes the human fibroblast growth factor receptor 3, and mutations

in this gene are found in both urothelial carcinoma [12, 13] and benign urothelial

papillomas [14, 15]. FGFR3 mutations occur mainly in the exons 7, 10, and 15, and the most common form is S249C (g. C746G) close to the 3’ end of the exon 7 [12–17].

These mutations are associated with lower grade and lower stage bladder cancers and

are thus considered a favorable prognostic marker [1, 16, 17]. Furthermore, a previous

study demonstrated a correlation between FGFR3 mutations and improved

progression-free survival (PFS) [17]. In contrast to their well-established role in

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urothelial carcinoma, evidence about FGFR3 mutations in PUNLMP is limited. One

recent study showed 60% of cases with mutated FGFR3 gene in a series of PUNLMP,

and no significant association with clinical outcome was found [11]. The value of

FGFR3 mutations in stratification of PUNLMP may need further investigation.

1.4.3 HRAS mutations

Mutations in another mitogen-activated protein kinase pathway-related gene,

HRAS, have been observed in some urothelial carcinomas [18, 19]. One large-scale

study showed an overall RAS mutation rate of 4% to 15% cases in MIBC depending on

the molecular subtype [20] (see section 1.5 for details). HRAS mutations are more

common in the benign inverted papillomas, as the percentage reached 81% (13/16) in

two separate studies [21, 22]. The most common form of HRAS mutations was Q61R,

which occurred in exon 3 of this gene [18, 21, 22]. Evidence about HRAS mutations is

other low-grade papillary urothelial neoplasms was more limited; the percentages of

mutation were 18% (2/11) and 3% (1/30) in urothelial papilloma and PUNLMP,

respectively, according to two separate studies [11, 22]. The association of HRAS

mutations and malignant potential in urothelial neoplasms is uncertain under current

evidence.

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1.5 Molecular classification of MIBC

While tumor recurrence is the main issue in management of low-grade papillary

urothelial neoplasms, MIBC could result in systemic metastasis and mortality. The

current treatment guidelines for MIBC are based on the tumor stage, overall clinical

condition, and the patient’s response to treatment [4]. In the recent decade, several

independent studies have revealed distinct molecular subtypes by analyzing mRNA

expression profiles in bladder UC specimens [23–26]. The concept of molecular

classification in bladder UC is similar to that in breast cancers [27, 28], and

immunohistochemical (IHC) staining for the breast cancer subtype-associated markers

has been incorporated in the treatment guidelines [29, 30]. Based on the achievements

in breast cancer studies, surrogate IHC markers for molecular subtypes of MIBC may

aid clinical management of the disease.

1.5.1 Lund and MDA Classification Systems

Before proceeding, we first take a brief review of the evolution in the molecular

classification systems. In 2012, a research group from Lund University (Sjödahl et al.)

revealed five distinct subtypes by analyzing the mRNA expression profiles in bladder

urothelial carcinomas. They named these subtypes as (1) urobasal A, (2) genomically

unstable, (3) infiltrated, (4) urobasal B, and (5) squamous cell carcinoma (SCC)-like,

according to their histologic or genetic characteristics [23]. Another group from MD

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Anderson Cancer Center (Choi et al.; abbreviated as MDA) described three subtypes

including (1) luminal, (2) basal, and (3) p53-like [24]. Along with the study by Cancer

Genome Atlas Research Network [25], each classification system could demonstrate the

significant association with the clinical outcome [23–25]. For example, the MDA basal

subtype is associated with a higher metastatic rate and shorter disease-specific survival

(DSS) [24]. However, the subtypes among these classification systems are not

interchangable because the gene sets for mRNA profiling are different. To be more confusing, the MDA “basal” subtype has a totally different meaning from the Lund

“urobasal” subtypes [23, 24, 31].

1.5.2 Consensus Molecular Classification System

Because of the diversity among these classification systems, researchers from different groups held a consensus meeting and described a “basal/squamous-like (BASQ)”

phenotype in the bladder urothelial carcinoma [31]. This BASQ phenotype is featured

by (1) high levels of cytokeratin (CK) 5/6 and CK14 expression, (2) low levels of

FOXA1 and GATA3 expression, (3) enrichment (higher percentage) of squamous

differentiation, and (4) worse clinical outcome. Compared to other subtypes, the BASQ

phenotype is more constantly defined among different classification systems; it is

roughly equivalent to the MDA basal subtype, the Lund SCC-like subtype, and the

TCGA cluster III and IV [31].

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After the consensus meeting, the researchers organized a multi-national study

using 1750 MIBC transcriptomes and analysis of independent molecular classification

systems [32]. The described a “consensus” set of six molecular classes: (1) luminal

papillary, which is enriched in noninvasive papillary components, (2) luminal

non-specified, (3) luminal unstable, (4) stroma-rich, (5) basal/squamous, which is

similar to the concept of BASQ phenotype, and (6) neuroendocrine-like, in the

descending order of overall survival (OS). These “consensus” subtypes differ in not

only histologic and clinical characteristics but also possibly underlying oncogenic

mechanisms [32].

1.5.3 Possible surrogate markers for molecular subtypes of MIBC

As stated in the previous section, four IHC markers are associated with the BASQ

phenotype described by Lerner et al.: CK5/6, CK14, FOXA1, and GATA3 [31]. Choi et

al. also reported consistency between the results of IHC staining and mRNA expression

profiles in basal (CK5/6-positive) and luminal (CK20-positive) subtypes [24]. Dadhania

et al. further proposed that IHC study on GATA3 and CK5/6 may sufficiently identify

these two subtypes with more than 90% accuracy [33]. Among these IHC markers,

GATA3, CK20, and CK5/6 are frequently used in the practice of diagnostic pathology.

The prognostic significance of GATA3 has been investigated in previous studies, but the

results were controversial. Miyamoto et al. revealed poor clinical outcome in MIBC

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with negative or decreased GATA3 expression [34], but three other studies

demonstrated no prognostic significance of GATA3 staining in bladder UC [35–37].

Similarly, negative GATA3 staining in upper-tract UC (UTUC) was associated with

poor clinical outcome in one study [38] but not in another [39]. The potential

importance of these IHC markers in MIBC warrants further investigation.

1.6 TP53 mutation and p53 IHC staining

In addition to subtype-specific genetic changes, TP53 mutation is common in bladder

urothelial carcinoma. In the practice of diagnostic pathology, IHC expression of p53 is

commonly used as a surrogate marker for TP53 mutations. This is based on the finding

that missense mutations in TP53 increase the half-life of p53, thereby increasing the

percentage of positive IHC staining for p53 [40–42]. Traditionally, a positive p53

staining is defined as nuclear reactivity over a certain cut-off percentage, whereas the

absence of p53 staining indicates a negative result [43, 44]. By using this criterion, the

correlation of p53 IHC staining with clinical outcome may be controversial [43].

Studies on ovarian and endometrial carcinoma have reported two patterns related to

TP53 mutation–associated aberrant p53 staining: diffusely strong nuclear reactivity and

complete absence of staining [45, 46]. The cut-offs for diffuse nuclear staining were

60% and 75% for ovarian [45] and endometrial [46] carcinoma, respectively. A bladder

cancer study also demonstrated that abnormal (negative or ≥50%) p53 staining was

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correlated with significantly worse recurrence-free survival (RFS) [44]. Therefore, the

clinical significance of p53 IHC staining in bladder cancers also requires further

investigation.

1.7 Aims of our study

In order to improve clinical management of bladder tumors, we investigated the

mutation status of the TERT promoter, FGFR3 gene, and HRAS gene in cases of

low-grade papillary urothelial neoplasms, including inverted papilloma, PUNLMP, and

low-grade NIPUC. We then analyzed the association between the mutation status and

the clinical outcome in these cases. In addition, we evaluated the IHC expression of

GATA3, CK20, CK5/6 and p53 in a series of MIBC cases and correlated them with the

associated clinical outcome, Ki-67 proliferative index, and other clinicopathological

parameters to investigate their clinical significance.

(24)

2. Materials and Methods

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

(25)

201508043RIND; revised on Dec 30^th, 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 16^th, 2017 (No.

201708055RIND; revised on Aug 17^th, 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

(26)

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

(27)

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

(28)

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).

(29)

3. Results

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

(30)

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

(31)

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

(32)

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

(33)

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

(34)

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

(35)

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.

(36)

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.

(37)

4. Discussion

4.1 Biological significance of TERT promoter mutation in papillary urothelial

neoplasm of low malignant potential

In human oncology, urothelial neoplasm is one of the neoplasms with well-established

pathogenetic models. At the genetic level, noninvasive papillary urothelial neoplasms

can be roughly divided into 2 groups: a genetically stable low-grade group and a

genetically unstable high-grade group. High-grade urothelial neoplasms are

characterized by TP53 mutations and exhibit aggressive biological behavior [2, 51–53].

By contrast, low-grade urothelial neoplasms are less likely to result in mortality.

According to the current WHO tumor classification, low-grade neoplasms include

totally benign urothelial papillomas (including exophytic papillomas and more common

inverted papillomas), PUNLMP, and low-grade NIPUC [2]. Although PUNLMP and

low-grade NIPUC are considered indolent tumors, they are not clinically insignificant

due to their frequent recurrence and the potential of progression to high-grade NIPUC

or invasive carcinoma. Between them, patients with low-grade NIPUC are more likely

to suffer from tumor recurrence than those with PUNLMP [3], and our results are

consistent with this general finding.

According to the current WHO criteria, classification of low-grade urothelial

(38)

genetic studies [2]. Some researchers have proposed the concept of “genetic grading”

for urothelial neoplasms [17, 54], but the complexity and cost of genetic studies may

hinder the feasibility of genetic grading in clinical practice. The results of our study

indicated that PUNLMP with wild-type TERT promoter had a lower recurrence rate. In

contrast, PUNLMP cases with TERT promoter mutations had significantly higher risks

of recurrence, which were similar to those in the low-grade NIPUC group. These results

suggest that PUNLMP cases can be stratified by TERT promoter mutation status for

prognostic purposes, and investigation of this mutation status may be of value for

clinical decision when treating patients with PUNLMP.

Compared with PUNLMP, our study did not show prognostic significance for

TERT promoter mutations in patients with low-grade NIPUC. Similarly, a study of

urothelial bladder cancers by Allory et al. found no association of the TERT promoter

mutation status with the clinical outcome [7]. Rachakonda et al. reported that TERT

promoter mutations are associated with poor survival and a higher recurrence rate in

bladder cancer patients without the common polymorphism rs2853669, which is located

in a preexisting Ets2 binding site in the TERT promoter. Such an association was not

found in patients with that polymorphism. The possible mechanism underlying the

association is that TERT promoter mutations affect the clinical outcome by creating de

novo Ets/TCF binding sites, and that common polymorphism may modify such effects

(39)

[55]. However, Rachakonda et al. did not include cases of PUNLMP, and further

investigation is necessary to explain the possible mechanisms of the effects of TERT

promoter mutations in PUNLMP.

Our results showed a high prevalence (76%) of HRAS mutation in inverted

papillomas. A previous study also showed HRAS mutations in 10 (91%) of 11 inverted

papillomas, and the authors postulated that isolated RAS mutations might induce cellular

senescence and result in a benign clinical course [22]. In our cohort, the three cases of

PUNLMP with HRAS mutations did not show coexistent TERT promoter or FGFR3

gene mutations. Two of them showed partially inverted growth patterns, and none of

these three tumors recurred after resection. Although these results were inconclusive due

to the limited case number, PUNLMPs with HRAS mutations might have similar

characteristics to the benign inverted papillomas. By contrast, two of the five low-grade

NIPUCs with mutated HRAS also showed TERT promoter mutation, and one additional

case had coexistent HRAS and FGFR3 mutations in our study. None of these tumors had

inverted growth in histology. The different biologic significance of HRAS mutations in

these low-grade urothelial neoplasms may worth further investigation.

Back to the issue of TERT promoter mutation, comparing our results with previous

results is potentially problematic. For survival analysis, many studies evaluating TERT

promoter mutations in bladder tumors have included cases with different grades or

(40)

stages [7, 8, 55]. In our study, the prognostic value of TERT promoter mutations fell to

an equivocal level if PUNLMP and low-grade NIPUC cases were mixed (p = .052).

Moreover, and probably more important, the actual cut-off points of histological

classification are subject to interobserver variation [56, 57]. In fact, all of the low-grade

NIPUC cases in our study were originally diagnosed as PUNLMP or even inverted

papilloma. Although our study found no TERT promoter mutation in any case of

inverted papilloma, Cheng et al. reported TERT promoter mutations in 15% (4/26) cases

of inverted papillomas [8]. Such discrepancy might result from the ethnic factor or other

causes; however, the potential interobserver variation may influence the accuracy of

metanalysis among these studies.

There are three potential limitations in our study. First, tumor progression was

uncommon in our study cohort and thereby limiting the analysis of PFS. Secondly, we

met technical problems about TERT promoter mutation analysis in three cases,

including one PUNLMP. This incident did not affect the statistical significance in our

study, but it might hamper the clinical application of TERT promoter mutation analysis.

Finally, to minimize the potential heterogeneity in our study cohort, we only selected

the primary (initial) specimen for each patient with tumor recurrence(s). The recurrent

tumors might exhibit different mutation patterns from their primary counterparts, but

investigations of such difference and possible clinical importance were beyond our

(41)

scope in this study.

4.2 Biological significance of GATA3, cytokeratin 20, cytokeratin 5/6 and p53

expression in MIBC

GATA3 is a transcription factor useful in histological diagnosis for UC [35, 58, 59]. It is

also recognized as a marker of luminal subtype(s) in the bladder cancer according to

recent research [20, 24, 33]. In this study, we noted that tumors with decreased GATA3

staining had significantly higher Ki-67 proliferative indices. In addition, IHC staining

for GATA3 was correlated with a clinical outcome in chemotherapy-naïve patients with

MIBC. Cases with diffuse GATA3 staining had the best outcome, and a minor

proportion (12.1%) of GATA3-negative tumors were prone to early recurrence with a

borderline trend of worse DSS. The prognostic significance of GATA3 was independent

to stage and nodal metastasis in RFS. These findings are compatible with the relatively

aggressive behavior observed in the BASQ tumors [24, 26, 32, 33].

The BASQ phenotype described by Lerner et al. included positive CK5/6 and

negative GATA3 in IHC staining [31]. However, we found it difficult to define the

actual BASQ subgroup in our study cohort. In this study, diffusely strong CK5/6

staining was not necessarily associated with negative GATA3. The association of these

two markers with the Ki-67 index might aid in understinaing this problem. Difference in

the Ki-67 index was significant at a 80% cut-off for GATA3, but the tumors with

(42)

negative and partial GATA3 staining had similar Ki-67 indices. Similar relationship was

observed between CK5/6 and Ki-67 index. If we use the 80% cut-off to define the

BASQ phenotype (CK5/6 and GATA3 staining in >80% and ≤80% of tumor cells,

respectively), this subgroup would account for 20 (22.0%) cases in our MIBC cohort.

Similar to GATA3 alone, the BASQ cases had a worse RFS (p = 0.027) compared with

others in the chemotherapy-naïve group. Moreover, CK5/6 alone was not prognostically

significant regarding RFS. From the view of prognostication, decrease in GATA3

expression may be a more important component than diffuse CK5/6 staining in the

BASQ phenotype.

Although GATA3 and CK5/6 demosntrated significantly negative correlation in

our study, staining for these two markers showed certain overlap in 44 (48.4%) MIBC

cases. Furthermore, diffuse coexpression was not rarely encountered (14.29%) in our

study. Such a diffuse coexpression phenomenon has not been well-described in previous

studies, and simultaneously high GATA3 and CK5/6 expression on protein level were

uncommon or even absent in the studies by Sjödahl [60] and Hodgson [36]. Dadhania et

al. showed some cases with overlap in GATA3 and CK5/6 staining; however, tumors

with >80% positivity for both markers were absent in their data [33]. A possible

explanation to this phenomenon is the ethnic difference. Further research based on

Asian population is warranted to confirm these findings.

(43)

The prognostic significance of GATA3 in bladder UC was controversial in

previous studies [34–37]. Miyamoto et al. reported that loss of GATA3 expression

predicted poor prognosis for patients with MIBC [34], but three other studies showed

that GATA3 expression had no significant influence on either DSS or RFS [35–37]. In

addition to ethnicity, three possible reasons can explain this discrepancy. First, we used

whole slides of the tumor specimens for IHC staining instead of tissue microarrays

(TMAs), which was the case in previous studies [35–37]. Partial staining might lead to

false-negative results in TMA, and this could potentially affect the significance in

survival analyses. Second, Kollberg et al. included 66 (17%) stage T1 tumors along

with MIBC [37], which may influence the results. Finally, in contrast to previous studies

[20, 32], the molecular subtypes in their cohort was not associated with clinical

outcomes [37]. The potential difference in the underlying population might result in

such discrepancy.

As CK20 and CK5/6 are well-established markers related to molecular subtypes

[24, 31, 33, 60], it may appear unreasonable that these markers did not have prognostic

significance. However, the molecular subtypes were defined through hierarchical

analysis with a large panel of markers. Previous studies revealed that the molecular

subtypes were associated with clinical outcomes [24, 31, 33, 60], but each marker

related to the subtypes was not necessarily significant in clinical outcomes. Kollberg et

(44)

al. also showed no prognostic significance in any single subtype-associated marker [37].

Therefore, the association of GATA3 expression and clinical outcome merits further

investigation to prove its value in clinical management.

The interpretation of p53 IHC staining is also noteworthy. Although we could not

find the prognostic significance of p53 staining, the differences in the Ki-67 index

suggested that using the same criteria as those used for ovarian carcinoma would be

more suitable when interpreting p53 staining in bladder cancer. Hodgson et al.

discovered that null staining of p53 should be considered an aberrant staining pattern

[44]. Our study further revealed the potential importance of intensity in the diffuse

nuclear staining group. Tumors with diffuse nulcear staining but variable intensity for

p53 (score 2) did not have higher Ki-67 indices than did the partial staining (score 1)

group. However, the differences in the variable-intensity (score 2) and strong-intensity

(score 3) groups were significant. Based on our findings, we plan to correlate these

criteria with the TP53 gene status and verify their superiority over the traditional

overexpression criteria for bladder cancer in the future.

(45)

5. Conclusion

In conclusion, the first part of our study provided the evidence that the TERT promoter

mutation is related to a higher risk of recurrence in PUNLMP cases. Such an association

was not observed in low-grade NIPUC, although the rates of the TERT promoter

mutation were similar between these two entities. Furthermore, the risk of recurrence in

PUNLMP cases with TERT promoter mutations was similar to that of low-grade NIPUC

cases. Mutation study of the TERT promoter may facilitate risk stratification and assist

the clinical decision when treating patients with PUNLMP of the urinary bladder.

In the second part of our study, decrease in GATA3 staining was significantly

associated with high proliferative activity and poor clinical outcome in MIBC. IHC

staining for GATA3 might facilitate in risk stratification in patients with MIBC

receiving radical cystectomy. Combining CK5/6 and GATA3 for prognostic

stratification has potential problems because coexpression is common. Defining the

“aberrant” p53 staining by using the criteria for ovarian carcinoma (complete

absence,strong nuclear reactivity in ≥60% of tumor cells, or diffuse cytoplasmic

staining) may be more suitable than the traditional overexpressionconcept; however, our

results did not demonstrate the direct association of p53 expression with clinical

outcome.

(46)

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