Novel liquid immunocytochemistry with machine learning analysis for bladder cancer detection

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 promoter mutation was associated with a higher recurrence rate in PUNLMP (p = 0.024) 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-naive 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 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.

Objective Papillary urothelial neoplasm of low malignant potential (PUNLMP) and stage TaG1 non-muscle invasive bladder cancer (NMIBC) represent separate categories in current WHO 1999 grade definitions. Similarly, PUNLMP and Ta low-grade are separate entities in the WHO 2004/2016 grading system. However, this classification is currently questioned by reports showing a similar risk of recurrence and progression for both categories. Patients and methods In this population-based study, risk estimates were evaluated in patients diagnosed with PUNLMP (n = 135) or stage TaG1 (n = 2176) NMIBC 2004–2008 with 5-year follow-up registration in the nation-wide Bladder Cancer Data Base Sweden (BladderBaSe). The risk of recurrence was assessed using multivariable Cox regression with adjustment for multiple confounders (age, gender, marital status, comorbidity, educational level, and health care region). Results At five years, 28/135 (21%) patients with PUNLMP and 922/2176 (42%) with TaG1 had local recurrence. The corresponding progression rates were 0.7% (1/135) and 4.0% (86/2176), respectively. A higher relative risk of recurrence was detected in patients with TaG1 tumours compared to PUNLMP (Hazard Ratio 1.6, 95% CI 1.2–2.0) at 5-year follow-up, while progression events were too few to compare. Conclusions The difference in risk of recurrence between primary stage TaG1 and PUNLMP stands in contrast to the recently adapted notion that treatment and follow-up strategies can be merged into one low-risk group of NMIBC.

Recurrent papillary urothelial neoplasm of low malignant potential. Subtle architectural disorder detected by quantitative analysis in DAXX-immunostained tissue sections

Although the papillary urothelial neoplasm of low malignant potential (PUNLMP) diagnostic category was retained in the updated 2016 World Health Organisation (WHO) classification of tumours of the urinary system, there still exists a great deal of controversy regarding the biological behaviour of these tumours. We review PUNLMP tumours and histological grading with an emphasis on the histomorphological, genetic and clinical similarities between PUNLMP and low-grade non-invasive papillary urothelial carcinoma. A literature search using PubMed was performed. All relevant literature concerning PUNLMP and the grading of urothelial tumours was reviewed. PUNLMPs cannot be reliably distinguished from low-grade non-invasive papillary urothelial carcinomas based on the histomorphological criteria outlined in the WHO 2004/2016 classification system. PUNLMPs and low-grade non-invasive papillary urothelial carcinomas are not only morphologically similar, but also share similar molecular genetic alterations and a similar risk of recurrence and progression. In addition, there are no consensus recommendations for a different method of treatment and follow-up for these two tumour types. Attempting to distinguish PUNLMP from low-grade papillary urothelial carcinoma adds an unnecessary level of complexity to the grading and classification of urothelial tumours. We feel that PUNLMP terminology should be abandoned and that all such tumours should be classified as low-grade carcinomas until more objective determinants of clinical outcome can be established.

Background: A preceding exploratory study (J. Clin. Pathol. 57(2004), 1201–1207) had shown that a karyometric assessment of nuclei from papillary urothelial neoplasms of low malignant potential (PUNLMP) revealed subtle differences in phenotype which correlated with recurrence of disease. Aim of the Study: To validate the results from the exploratory study on a larger sample size. Materials: 93 karyometric features were analyzed on haematoxylin and eosin-stained sections from 85 cases of PUNLMP. 45 cases were from patients who had a solitary PUNLMP lesion and were disease-free during a follow-up period of at least 8 years. The other 40 were from patients with a unifocal PUNLMP, with one or more recurrences in the follow-up. A combination of the previously defined classification functions together with a new P-index derived classification method was used in an attempt to classify cases and identify a biomarker of recurrence in PUNLMP lesions. Results: Validation was pursued by a number of separate approaches. First, the exact procedure from the exploratory study was applied to the large validation set. Second, since the discriminant function 2 of the exploratory study had been based on a small sample size, a new discriminant function was derived. The case classification showed a correct classification of 61% for non-recurrent and 74% for recurrent cases, respectively. Greater success was obtained by applying unsupervised learning technologies to take advantage of phenotypical composition (correct classification of 92%). This approach was validated by dividing the data into training and test sets with 2/3 of the cases assigned to the training sets, and 1/3 to the test sets, on a rotating basis, and validation of the classification rate was thus tested on three separate data sets by a leave-k-out process. The average correct classification was 92.8% (training set) and 84.6% (test set). Conclusions: Our validation study detected subvisual differences in chromatin organization state between non-recurrent and recurrent PUNLMP, thus allowing a very stable method of predicting recurrence of papillary urothelial neoplasms of low malignant potential by karyometry.

Objective To investigate the mRNA expression of Enhancer of zeste homolog 2 (EZH2)and B-cell-specific moloney murine leukemia vires insertion site 1(Bmi-1)in transitional cell carcinoma of the bladder tissue and their roles in the development and progression of bladder carcinoma.Methods The mRNA expression of EZH2 and Bmi-1 in 70 cases of the bladder carcinoma tissue and 15 cases of normal bladder mucosa waft detected by RT-PCR and real-time quantitative PCR.The bladder cancer cell line,T24 was used as a positive control.Results The expression of EZH2 and Bmi-1 wad undetectable or weak in normal bladder mucosa.The mRNA expression levels of EZH2 and Bmi-1 in the bladder carcinoma were higher than in the normal control group(P＜0.01).The mRNA expression of EZH2 in papillary urothelial neoplasms of low malignant potential(PUNLMP)group,low-grade group and high-grade group was 0.1082±0.1880,0.0740±0.0984 and 0.1885±0.2144 respectively,and that in normal control group was 0.0158±0.0208(P＞0.05,＜0.01 and＜0.01 respectively).It was higher in invagive group(0.2069±0.2241)than in superficial group(0.0868±0.1233)(P＜0.01).The mRNA expression of Bmi-1 in PUNLMP group,low-grade group and high-grade group wag 0.6654±0.7668,0.4955±0.4553 and 0.7986±0.9259,and that in normal control group wag(0.1745±0.0973)(P＞0.05,＞0.05 and＜0.01 respectively).The mRNA expression of Bmi-1 in invagive group(0.8368±1.0041)wag higher than in superficial group(0.5505±0.4992)(P＞0.05).Conclusion The expression of both EZH2 and Bmi-1 genes may play an important role in the occurrence of bladder carcinoma,and EZH2 is more closely correlated with the occurrence and progression of bladder carcinoma. Key words: Transitional cell carcinoma of the bladder; Enhancer of zeste homolog 2 gene; Bcell-specific moloney murine leukemia virus insertion site 1 gene; Real time quantitative PCRcell-specific moloney murine leukemia virus insertion site 1 gene; Real time quantita

Mutations in FGFR3 and the promoter region of the telomerase reverse transcriptase (TERT) gene have been found frequently in urothelial carcinoma of the urinary bladder. However, related data for papillary urothelial neoplasm of low malignant potential (PUNLMP) are limited. In this study, we investigated the mutation status of the TERT promoter, FGFR3 and HRAS in low-grade papillary urothelial neoplasms and evaluated their prognostic significance. The cases included in this study comprised 21 inverted papillomas, 30 PUNLMPs and 34 low-grade non-invasive 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 observed more frequently 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 promoter mutation was associated with a higher recurrence rate in PUNLMP (P=0.024) 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.

Objectives To explore the clinical significance of tyrosine kinase receptor RON mRNA expression and it's splicing variant in bladder tumors. Methods Sixty-three cases of transitional cell carcinoma of the bladder (TCCB), including 30 cases of pathological grade I , 15 cases of pathological grade Ⅱ and 18 cases of pathological grade Ⅲ (44 cases of clinical stage Tis + T1, 15 cases of T2 + T3 +T4), 7 inverted papilloma of the bladder ( IPB), 9 cases of papillary urothelial neoplasm of low malignant potential (PUNLMP) and 12 cases of normalbladder mucosa RT-PCR was employed with the internal standard (GAPDHmRNA) to detect the expression of RON mRNA. PCR and direct sequencing was then utilized to identify the potential RON mRNA splicing variants. Finally, the variants' positive rates of expression were analyzed among the different tissues, diverse TCCB pathological grades and clinical stages. Results The expression levels of RON mRNA/GAPDH mRNA among TCCB, IPB, PUNLMP and normal control were 4. 9 × 10-3 ( 1. 8 × 10-3-1.0 × 10-2 ), 3. 8 × 10-3 (2. 4 × 10-3-1.7 × 10-2 ), 4. 9 ×10 -3 ( 1.7 × 10 -3-1.1 × 10 -2 ) and 1.0 × 10-3 (4. 5 × 10-4-2. 8 × 10-3 ) respectively. The difference had a statistical significance (x2K-W = 17. 278 ,P ＜0. 05 ). The expression levels among pathological grade I, Ⅱ and Ⅲ were 3.7 × 10-3( 1.3 × 10-3-7.5 × 10-3) , 4. 9 × 10-3(1.9 × 10-3-1.1 × 10-2) and 8.9 × 10-3(2. 7 ×10 -3-8.0 × 10 -2 ) respectively. The erpression levels among the clinical stage Tis + T1 and T2 + T3 + T4were 3.5 × 10-3 ( 1.2 × 10 -3-7. 7 × 10-3 ) and 9. 7 × 10 -3 ( 2. 9 × 10-3-5. 3 × 10-2 ). The differences between expression levels were of statistical significance among the different pathological grades ( x2k-W =7. 341, P ＜0. 05 ) and clinical stages ( Z = - 2. 306, P ＜ 0. 05 ). The positive rates of exon 11deletion(E11△) in TCCB, IPB and PUNLMP were 71％ (45/63), 57％ (4/7) and 67％ (6/9) respectively, andthe total positive rate in bladder tumor tissues was 70％. Meanwhile, expression of the novel RON variant wesnot detected in the normalbladder mucosa. The positive expression rate of E1 1△ has no significant correlationamong the different clinical pathological tissues (x2 = 0. 620, P ＞ 0. 05 ). There was no statistical significancein expression positive rate between different pathological grades ( Z =0. 221, P ＞0. 05 ) and clinical stages( Z = 0. 538, P ＞ 0. 05) as well. A novel RON splice variant, deletion of RON exon 11 3 476 - 3 539 ( E3476 -3539△) was fond in the pathological tissue. The positive expression rates of E3 476 -3 539 in TCCB,IPB and PUNLMP were 57％ (36/63), 43％ (3/7) and 56％ (5/9) respectively, and the total positive expression rate was 56％ (44/79). The positive rates of E3 476 -3 539△ in pathological grade I , Ⅱ and Ⅲ were 40％ ( 12/30), 67％ (10/15) and 78％ (14/18), and it's positive rates in clinical stage Tis +T1and T2 +T3 + T4 were 48％ (21/44) and 80％ (12/15). The differences in each group had significantly statistical significance ( Z = 7. 285, 5. 041, P ＜ 0. 05 ) . However, the positive rates amongdifferent pathological tissues had no significance (x2 = 0. 517, P ＞ 0. 05 ). Conclusions The expression level of RON mRNA is significantly associated with histological grading and clinical stage. RON may play an important role in the progression ofTCCB. Compared with the normal control, the increased RON variant expression may contribute to the carcinogenesis of the bladder tumor. Key words: Urinary bladder neoplasms; Receptor protein-tyrosine kinases; Alternative splicing

In the 1999 World Health Organization classification system, papillary tumors of the urinary bladder were classified as papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and as Grade 1, Grade 2, and Grade 3 urothelial carcinoma. The biologic potential of PUNLMP of the urinary bladder is controversial. To the authors' knowledge, information regarding the genetic changes of PUNLMP tumors of the bladder is limited. The authors examined loss of heterogygosity (LOH) at 5 polymorphic microsatellite markers on chromosome 9q32-33 (D9S177), 9p22 (IFNA), 17p13.1 (TP53), 12q14-24 (D12S1051), and 3p25-26 (D3S3050) from 26 patients who were diagnosed with PUNLMP tumors of the urinary bladder. Tumors were microdissected from sections prepared from formalin-fixed, paraffin-processed tissue specimens using laser capture microdissection. LOH was found in 21 of 26 (81%) patients with PUNLMP. The rate of LOH was 41% with D9S177, 32% with IFNA, 29% with TP53, 26% with D12S1051, and 44% with D3S3050. Allelic loss of multiple chromosome loci was often present in patients with PUNLMP tumors. Genetic changes that commonly occur in advanced bladder carcinoma (> or = pT2) are frequently found in PUNLMP of the urinary bladder.

Papillary urothelial neoplasm of low malignant potential (PUN-LMP): Still a meaningful histo-pathological grade category for Ta, noninvasive bladder tumors in 2019?

We investigate the distribution of bladder tumor category and stage in Northern New England by geographic region, smoking status and over time. 1091 incident bladder cancer cases from the New England Bladder Cancer Study (NEBCS), a large population-based case-control study carried out in Maine, New Hampshire and Vermont (2001-2004), and 680 bladder cancer cases from previous case-control studies in New Hampshire (1994-2000) were used in the analysis. Of 1091 incident bladder cancer cases from the NEBCS, 26.7% of tumors were papillary urothelial neoplasms of low malignant potential (PUNLMP), 26.8% low-grade papillary urothelial carcinomas (PUC-LG), 31.3% high-grade papillary urothelial carcinomas (PUC-HG), 9.1% non-papillary urothelial carcinomas (non-PUC), and 4.3% carcinoma in situ (CIS). Approximately 70% of cases were non-invasive (Tis/Ta), and all PUNLMP cases were of the Ta category. By contrast, half of all PUC-HG carcinomas were invasive. Short-term time trend analysis within the NEBCS (2001-2004) indicated an increase in the percentage of PUNLMP (p-trend<0.0001) paralleled by a decrease in PUC-LG (p-trend=0.02), and for PUC-LG an increase in the percentage of non-invasive tumors (p-trend 0.04). Our findings suggest possible short-term trends with an increase in the percentage of PUNLMP and a change in the percentage of PUC-LG towards non-invasive disease.

Inverted lesions in the urinary bladder have been the source of some difficulty in urological pathology. The two common ones are von Brunn's nests and cystitis cystic/cystitis glandularis, which are considered normal variants of urothelium. Apart from them, a number of other rare urothelial lesions with inverted growth pattern occur in the urinary bladder. Some of them are only reactive conditions, just as pseudocarcinomatous hyperplasia. Some are benign tumors, namely inverted papilloma. Whereas others are malignant neoplasms, including inverted papillary urothelial neoplasm of low malignant potential (PUNLMP), non-invasive inverted papillary urothelial carcinoma (low-grade and high-grade), and invasive urothelial carcinoma (inverted, nested and big nested variants). Because of the overlapping morphological features of all the inverted lesions mentioned above, even between high-grade invasive carcinoma and pseudocarcinomatous hyperplasia which are only a kind of reactive conditions, it is very important for the surgical pathologist to recognize and be familiar with these inverted lesions in urinary bladder. In this article, we review these spectrums of inverted lesions of the urinary bladder. Emphasis is placed on histogenesis, morphology, differential diagnosis of these lesions, and the pathologic grading of the non-invasive inverted neoplasms, such as inverted papilloma, inverted PUNLMP, non-invasive inverted papillary urothelial carcinoma with low-grade, and non-invasive inverted papillary urothelial carcinoma with high-grade.

Papillary urothelial neoplasm of low malignant potential (PUNLMP) is a clinically significant lesion because recurrence occurs in approximately 35% of patients. To date, it is not possible to identify those cases that will recur based on conventional histopathologic assessment. The objective of the current study was to evaluate immunohistochemically tissue expression of fibroblast growth factor receptor 3 (FGFR3), cytokeratin 20 (CK20), and MIB-1 in nonrecurrent and recurrent PUNLMP. FGFR3, CK20, and MIB-1 were investigated by immunohistochemistry (IHC) in 80 PUNLMP cases (41 nonrecurrent and 39 recurrent), in 4 cases of normal urothelium (NU), and in 5 cases of muscle invasive pT2 urothelial carcinoma (UC). Statistics included discriminant analysis. NU demonstrated a weak to moderate FRFG3 staining intensity, a superficial pattern of CK20 staining, and low proliferative activity. UC was found to have FGFR3 staining similar to NU, an abnormal CK20 expression, and high proliferative activity. The nonrecurrent PUNLMP group demonstrated strong FGFR3 intensity in 80.5% of the cases (vs 56.4% of the recurrent cases), a superficial CK20 staining pattern in 53.7% of the cases (vs 28.2% of the recurrent cases), and a percentage of MIB-1-positive nuclei below the median value of all the PUNLMP cases in 61% of the cases (35.9% in the recurrent cases). The differences were statistically significant. Discriminant analysis based on these 3 features demonstrated that 67.5% of cross-validated grouped PUNLMP cases were correctly allocated, with 73.2% of the nonrecurrent and 61.5% of the nonrecurrent cases being correctly classified. The specificity and sensitivity were 73.1% and 61.5%, respectively. Strong immunohistochemical expression of FGFR3, a superficial staining pattern of CK20, and a low proliferative activity define those papillary urothelial neoplasms of low malignant potential that do not recur.

Aim: To analyse nuclear chromatin texture in non-recurrent and recurrent papillary urothelial neoplasms of low malignant potential (PUNLMPs). Materials: Ninety three karyometric features were analysed on haematoxylin and eosin stained...

Purpose: To verify if the distinction between papillary urothelial neoplasm of low malignant potential (PUNLMP) and noninvasive low-grade papillary urothelial carcinoma (LGPUC) reflects a different biologic activity.Materials and Methods: We reviewed and analyzed the clinical data from 678 patients who had a diagnosis of PUNLMP (n=53) or noninvasive LGPUC (n=625) after initial TUR-BT for bladder neoplasm between 2000 and 2012.Results: The noninvasive LGPUC group showed a higher frequency of recurrence in comparison with the PUNLMP group (46.7% vs. 30.2%, p=0.022). In contrast, there were no significant differences in progression (15.2% vs. 18.9%, p=0.295) between the two groups. Grade progression was reported in 10 patients (LG: n=5; high grade: n=2; carcinoma in situ: n=3) and stage progression was reported in 2 patients (all: T1) in PUNLMP group. The Kaplan-Meier survival analysis showed significantly decreased 5-year recurrence-free survival (RFS) (50.3% vs. 74.6%, log-rank test, p=0.014) in the noninvasive LGPUC group compared to the PUNLMP group. However, there were no significant differences in progression-free survival (PFS) between the two groups. Multivariate analysis revealed that tumor grades according to 2004 WHO/ISUP classification system (PUNLMP vs. LG) were identified as significant predictors of RFS. However, it was not a significant predictor of both PFS and overall survival.Conclusions: PUNLMP had a substantial number of recurrences (30.2%), although RFS was better than noninvasive LGPUC. In addition, PUNLMP had a similar risk of progression compared with noninvasive LGPUC. Consequently, PUNLMP should be treated in a manner similar to noninvasive LGPUC, and long-term clinical follow-up should be recommended for patients with PUNLMP.