KDM6A expression loss is frequent in low grade non-invasive urothelial carcinomas of the urinary bladder

Objective. The gene lysine demethylase 6A (KDM6A) located on chromosome Xp11 often shows truncating mutations in urothelial carcinoma. Mutations resulting in protein expression loss can be detected by immunohistochemistry (IHC).
Methods. A tissue microarray with >2,500 bladder tumors was analyzed by IHC. 78 cancers were sequenced for KDM6A.
Results. KDM6A expression loss decreased from 36% of 345 pTaG2 low-grade to 23% of 152 pTaG2 high-grade and 18.5% of 92 pTaG3 tumors (p=0.0004) but not further in pT2-4 cancers (17.2-21.9%). KDM6A staining was unrelated to pT, pN, grade, and overall survival (p>0.1894) in 636 patients with pT2-4 cancers. KDM6A loss was more common in male (22.2%) than in female patients (15.4%; p=0.0067), and in tumors from males with Y-chromosome loss (36.1%) than without Y-loss (16.3%; p<0.0001). A KDM6A loss occurred in all 15 male and in 17 (74%) of 23 female patients with a truncating KDM6A mutation, but only 15 (75%) of 20 male and 17 (81%) of 21 female patients with KDM6A expression loss had a truncating mutation.
Conclusions. KDM6A expression loss is frequent in urothelial carcinoma and mostly due to truncating mutations. KDM6A IHC may be a useful tool for the distinction of neoplastic from non-neoplastic urothelial cells in follow-up examinations of patients with KDM6A deficient cancers.

Introduction

Carcinoma of the urinary bladder is one of the most common malignant tumor types worldwide. About 80% of patients present with non-invasive (pTa) or minimally invasive (pT1) cancers which can be removed by transurethral resection. Most patients have a good prognosis, but at least 20% of pT1 cancers will further progress to muscle-invasive disease. At this stage, radical cystectomy (RC) is the surgical standard of care although distant metastasis develops in up to 50% of patients, most of whom eventually die from their disease. Significant shortcomings for early detection and appropriate individual treatment of bladder cancer include a low sensitivity of urine cytology ¹, limitations in the assessment of cancer aggressiveness ², and the lack of efficient treatment in case of systemic disease ³. It is hoped that our increased understanding of the molecular changes underlying bladder cancer development and progression will eventually lead to improved patient management.

Inactivating mutations of lysine demethylase 6A (KDM6A) belong to the most commonly found molecular aberrations in urothelial carcinomas, occurring in 30-52% of pTa and in 23-25% of pT2-4 cancers ^4,5. KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator that represents a critical part of the COMPASS-like complex. This protein complex can demethylate histone H3 at its 27th amino acid (H3K27me2/3) and thus defines a repressive histone modification that leads to transcriptional inactivation of various genes. Both, tumor suppressive and oncogenic roles of KDM6A have been proposed in cancer ⁶. In a study on bladder cancer cell lines, KDM6A loss resulted in a dependency on enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, and EZH2 inhibition resulted in decreased growth of KDM6A-null patient-derived cell line xenografts ⁷. Large scale studies on the role of KDM6A protein expression in bladder cancer are so far lacking. However, because of the fact that most KDM6A mutations are truncating, that KDM6A localizes to chromosome Xp11.3, and that due to imprinting not only males but also females should have only one active copy of the KDM6A gene, many KDM6A mutations must result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC).

To study the prevalence and the potential diagnostic and prognostic role of KDM6A expression loss, a previously collected cohort of more than 2,700 urothelial bladder carcinomas was analyzed by IHC in a tissue microarray (TMA) format, and the results were compared with histopathological parameters of cancer aggressiveness, patient outcome, KDM6A sequencing data, and previously collected data on the Y chromosome status of our cohort.

Materials and methods

TISSUE MICROARRAYS (TMAS)

Our set of TMAs contained one sample each from 2,710 urothelial tumors of the bladder archived at the Institute of Pathology, University Hospital Hamburg, Germany, Institute of Pathology, Charité Berlin, Germany, Department of Pathology, Academic Hospital Fuerth, Germany, or Department of Pathology, Helios Hospital Bad Saarow, Germany, and/or treated at Department of Urology, University Hospital Hamburg, Germany, Department of Urology, Charité Berlin, Germany, Department of Urology, Helios Hospital Bad Saarow, Germany, Department of Urology, Albertinen Hospital, Hamburg, Germany, and Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland. Patients at each center were treated according to the guidelines at the time. In brief, patients with pTa/pT1 disease underwent a transurethral resection of the bladder tumor with or without adjuvant instillation therapy, while most patients with pT2-4 disease were treated by radical cystectomy. Available histopathological data including grade, tumor stage (pT), lymph node status (pN), and status of blood vessel (V) and lymphatic vessel (L) infiltration are shown in Table I. The grading of pTa tumors included both a classification according to WHO 2004 which were valid at the time of the respective diagnoses. A centralized review of the cases was not done. Clinical follow-up data (overall survival; OS) were available from 636 patients with pT2-4 carcinomas treated by cystectomy. The tissues were fixed in 4% buffered formalin and then embedded in paraffin. For TMA manufacturing, one tissue spot (diameter: 0.6 mm) was transmitted from a cancer containing donor block into an empty recipient paraffin block. Conventional whole sections were prepared from donor tissue blocks of all 6 cancers from females with truncating KDM6A mutations but retained KDM6A staining in the TMA spots. Data on Y-chromosome loss was expanded from an earlier study ⁸. The use of archived remnants of diagnostic tissues for TMA manufacturing, their analysis for research purposes, and patient data were according to local laws (HmbKHG, §12) and the analysis was approved by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work was carried out in compliance with the Helsinki Declaration.

IMMUNOHISTOCHEMISTRY (IHC)

Freshly prepared TMA sections were immunostained on one day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 target retrieval solution (Agilent, CA, USA; #S2367). Endogenous peroxidase activity was blocked with peroxidase blocking solution (Agilent, CA, USA; #52023) for 10 min. Primary antibody specific for KDM6A (rabbit recombinant monoclonal, clone HMV-311, cat. # 2082-267M, ardoci GmbH, Hamburg, Germany) was applied at 37°C for 60 min at a dilution of 1:150. Primary antibody specific for Ki-67 (rabbit recombinant monoclonal, clone MSVA-267M, cat. # 2843-U3223, MS Validated antibodies GmbH, Hamburg, Germany) was applied at 37°C for 60 min at a dilution of 1:150. Bound antibody was then visualized using the EnVision kit (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with haemalaun. For KDM6A, the average staining intensity of unequivocally neoplastic cells was estimated as 0, 1+, 2+, 3+. For the classification of a tumor as negative (0), presence of unequivocal KDM6A staining in tumor adjacent normal cells was required. Tumors with complete absence of KDM6A staining in cancer cells but a lack of stromal cells with unequivocal KDM6A positivity were considered “non-informative”. For Ki-67, the proportion of tumor cells with nuclear staining was recorded.

KDM6A SEQUENCING

A total of 20 pTa tumors from males with a KDM6A expression loss, 21 pTa-pT4 tumors from females with a KDM6A expression loss and 37 pTa-pT4 tumors (16 male, 21 female) with retained KDM6A expression were subjected to sequencing. DNA was extracted from FFPE specimens using the Maxwell® RSC DNA FFPE Kit (Promega Corporation, Madison, WI, USA) and the Maxwell RSC 48 Instrument according to the manufacturer’s instructions. Targeted gene sequencing was performed on a MiniSeq (Illumina, San Diego, CA, USA) platform with a custom primer panel that covered all exons of the KDM6A gene (for details see Supplementary Tab. I). The DNA libraries were prepared using the AmpliSeq Library PLUS kit and Illumina CD indexes set B (Illumina, San Diego, CA, USA). Sample preparation was carried out in a PCR-based workflow following Illumina’s AmpliSeq protocol. In brief, 100-200 ng DNA were used in the PCR reaction as follows. Step 1: 1x at 99°C for 2 min, step 2: 20x at 99°C for 15 sec followed by 60°C for 4 min, step 3: 1x at 10°C hold) followed by partial digestion of the amplicons, ligation of indexes, clean up, amplification of the library, and second clean up. Libraries were sequenced on an Illumina MiniSeq according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). The sequencing data were analyzed using the CLC software package CLC Genomics Workbench v.22.0.2 (Qiagen, Aarhus A/S).

STATISTICS

Statistical calculations were performed with JMP 16 software (SAS Institute Inc., NC, USA). Contingency tables and the chi²-test were performed to search for associations between KDM6A expression loss, tumor phenotype, and molecular parameters. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups.

Results

TECHNICAL ISSUES

Of 2,710 urothelial cancer samples, 2,125 (78.4%) were interpretable for KDM6A IHC. Reasons for non-informative cases included absence of KDM6A positive stroma cells or of unequivocal cancer cells in the TMA spot, as well a complete lack of tissue spots.

KDM6A IMMUNOSTAINING

A distinct nuclear KDM6A staining was always seen in normal urothelium although it tended to be less intense in umbrella cells. KDM6A immunostaining was absent in 484 (22.8%), low (1+) in 371 (17.5%), moderate (2+) in 494 (23.2%), and strong (3+) in 776 (36.5%) of 2,125 analyzable urothelial bladder carcinomas. Examples of KDM6A stained tumor and normal urothelium samples are shown in Figure 1. As there were no significant differences between tumors with weak (1+), moderate (2+), and strong (3+) KDM6A positivity, all tumors with KDM6A positivity were grouped together as “KDM6A retained”. A complete KDM6A expression loss occurred in 36% of 350 pTa G2 low-grade, 23% of 152 pTa G2 high-grade, and 18.5% of 92 pTa G3 tumors (p=0.0004). As compared to pTa G3 tumors, the frequency of KDM6A expression loss did not differ significantly in pT2 (17.2%), pT3 (21.9%), and pT4 (18.2%) cancers. Within pT2-4 carcinomas, KDM6A staining was unrelated to pT, pN, grade, L-status, V-status, and overall survival (Tab. II, Fig. 2a; p>0.1894). KDM6A loss was significantly related to low tumor cell proliferation as measured by Ki-67 IHC. The Ki-67 labeling index (Ki-67 LI) was 12.5±0.7 in 422 tumors with KDM6A loss compared to 17.3±0.4 in 1467 tumors with retained KDM6A expression (p<0.0001). This relationship was independent of the tumor stage (pTa: p=0.0627, pT1: p=0.013, pT2: p=0.0402, pT3: p=0.0004, pT4: p=0.0256, see Supplementary Fig. 1). KDM6A loss was more common in male (22.2%) than in female patients (15.4%; p=0.0067) and within male patients, and was more frequent in tumors with Y-chromosome loss (36.1%) than in cancers without Y-chromosome loss (16.3%; p<0.0001). This relationship was also retained within pTa tumors, where a KDM6A loss occurred in 31.8% of male but in only 20.8% of female patients (p=0.0239), and – in males – a KDM6A loss was seen in 57.8% of tumors with Y-chromosome loss but in only 25.4% of cancers without a Y-chromosome loss (p<0.0001, Tab. III). The absence of a prognostic impact of KDM6 expression loss was independent of whether the Y-chromosome was lost or not in muscle invasive cancers (Fig. 2b).

KDM6A MUTATION ANALYSIS

Data on both KDM6A mutation and KDM6A expression were available on 78 tumors (Tab. IV). Truncating KDM6A mutations were seen in 48.7% and non-truncating KDM6A mutations in 5.1% of these selected cases. Truncating but not non-truncating KDM6A mutations were significantly associated with KDM6A expression loss (p<0.0001). This also held true for the subsets of 36 male (p<0.0001) and 42 female (p=0.0011) patients. All male patients with a truncating KDM6A mutation had complete KDM6 expression loss. There was, however, a small subset of tumors with a complete KDM6 expression loss that lacked truncating or even non-truncating KDM6A mutations both in male (n=5, 25%) and in female (n=4, 19%) patients. Images from these tumors with KDM6A expression loss in the absence of truncating KDM6A mutations are shown in Figure 3. In female patients, only 17 of 23 (73.9%) cancers with a truncating KDM6A mutation had complete KDM6A expression loss in the TMA spots. KDM6A IHC on the entire whole sections from the blocks used for sequencing of the remaining 6 cancers revealed a focal (heterogeneous) KDM6A staining loss in one of these tumors (Figs. 4a-b), a reduced but still significant KDM6A staining in at least half of the tumor tissue in 3 cancers (Fig. 4c), and normal (high-level) KDM6A expression in 2 tumors (Fig. 4d).

Discussion

Our data show that KDM6A expression loss is a common event in urothelial neoplasms, especially in low grade cancers. While IHC data on KDM6A in urothelial cancer are so far lacking, our rates of KDM6A loss of 36% in pTa G2 low-grade, 23% in pTa G2 high-grade, 18.5% pTa G3, and 20% in pT2-4 cancers is in line with earlier data using next generation sequencing. These have also found a predilection of KDM6A alterations in low grade and non-invasive cancers and described KDM6A mutations in 40-52% of low-grade pTa, 30-38% of high-grade pTa, and in 23-25% of pT2-4 cancers ^4,5. The somewhat higher rate of KDM6A mutations compared to KDM6A expression loss is obviously because only deleterious (truncating) KDM6A mutations can be detected by IHC as “expression loss”. In urothelial neoplasms, these made up for 69-80% of KDM6A mutations in males and for 62-82% in females in earlier studies ^9-13.

Some functional studies had earlier suggested that KDM6A loss would result in cellular dedifferentiation ¹⁴, epithelial-mesenchymal transition ¹⁵, enhanced cell motility and invasiveness ¹⁶, high tumor burden and reduced survival ¹⁷, increased tumor aggressiveness ¹⁸, and loss of luminal tumor phenotype ¹⁴ of urothelial cancer cells. Considering the strong association with low grade and non-invasive cancer and with a low tumor cell proliferation index, as well as the complete lack of a prognostic role and of significant associations with parameters of aggressive disease in muscle-invasive cancers, our data do not support a role of KDM6A dysfunction for cancer progression in vivo. Our data suggest, however, a significant clinical utility of KDM6A IHC for the distinction of urothelial neoplasms from non-neoplastic urothelial cells. The identification of low-grade urothelial cancer - the subgroup with the highest rate of KDM6A expression loss - is challenging by morphology alone. This especially applies to flat lesions, small and crushed biopsies, and cytological specimens. KDM6A IHC can therefore complement more established markers to enable a safe diagnosis of low-grade urothelial neoplasms, at least in the subsets of patients with deficient KDM6A expression. Frequently used markers for this purpose, including CK20, p53, CD44, and (increasingly) MTAP ¹⁹, share the disadvantages of either a low prevalence in low-grade neoplasms or a lack of an unequivocal tumor association. Therefore, KDM6A IHC appears to be a much more appealing test, since KDM6A loss is directly associated with a cancer-causing genomic event.

That KDM6A, considered a major bladder cancer driver gene, is located on X chromosome is of particular interest. The potential disbalance of KDM6A redundancy between male and female patients has been proposed as a possible cause for the 3-fold higher prevalence of bladder cancer in males than in females ¹⁷. In males, a catastrophic effect of a loss of X chromosome genes is often compensated by paralogs on the Y chromosome. Ubiquitous transcribed tetratricopeptide repeat containing Y-linked (UTY; KDM6C) is the KDM6A paralog on the Y chromosome. Similar as KDM6A, KDM6C can be incorporated into the COMPASS complex but has a slightly different function and lacks the demethylating properties of KDM6A ²⁰. That tumors with KDM6A loss had almost twice as many Y chromosome losses than tumors with retained KDM6A expression, both in our study and in a previous analysis by Ahn et al. ²¹, might suggest that the loss of both paralogs could result in an additional growth advantage of tumor cells. However, additional Y chromosome losses did not affect prognosis in tumors with a KDM6A expression loss in our study.

To assure similar expression levels as in males, one copy of X chromosome genes is normally repressed by imprinting in females. However, the imprinting mechanism is not 100% perfect and KDM6A is a gene that can escape X inactivation ²². The significant but not perfect link between KDM6A expression loss and truncating KDM6A mutations in females is consistent with a significant but incomplete imprinting of KDM6A. That 17 of 23 cases with a truncating KDM6A mutation in females showed a complete KDM6A expression loss demonstrates that a loss of the active KDM6A allele cannot be compensated by the second allele in the majority of cases. Most likely, this is due to efficient imprinting of the second KDM6A allele in these cases. Since 5 of the 23 female patients with deleterious KDM6A mutation showed only a focally reduced or even “normal” KDM6A expression indicates, however, the second KDM6A allele is either not or only incompletely inactivated by imprinting and can compensate for the destructed allele in a minor fraction of cases. It is unlikely that the KDM6A mutation affected the inactive/imprinted gene copy as this would hardly result in a growth advantage of affected cells.

It was expected that all 15 tumors from male patients with truncating KDM6A mutations had a complete KDM6A expression loss which provides a strong indirect confirmation of our IHC assay. The fact that 25% of 20 male and 19% of 21 female cancers with complete KDM6A expression loss did not show a truncating KDM6A mutation demonstrates that at least one other mechanism can cause a KDM6A expression loss. Possible mechanisms with such an effect include mutation or methylation of the promoter region, histone modification and chromatin remodeling, transcriptional or post-transcriptional gene silencing by siRNAs, miRNAs, lncRNAs or other RNA binding proteins, genomic deletion, or combinations thereof ^23-25. A query of the cBioportal database for cancer genomics ^26-28 including 11 studies ^{5,11,12,29-36} revealed that deep or shallow deletions (largely corresponding to homo- or heterozygous deletions) of the KDM6A locus can be found in 1% (deep deletion) and 8% (shallow deletion) of female bladder cancer patients lacking truncating KDM6A mutations. Irrespective of the mechanisms leading to KDM6A deficiency, tumors with a KDM6A expression loss may be candidates for treatment with EZH2 inhibitors ³⁷. KDM6A and EZH2 have opposing roles in regulating H3K27 methylation ³⁸. In KDM6A-mutant tumors, dysregulated EZH2 activity leads to excessive H3K27 methylation and transcription repression ³⁹, causing tumor suppressor gene silencing, cancer initiation and progression ⁴⁰. There is evidence that inhibiting EZH2 in KDM6A-mutant cancers can restore histone methylation balance and reactivate tumor suppressor genes such as IGFBP3 ⁷ or IRF1 ⁴¹. Functional analyses have shown that EZH2 inhibition results in decreased cell growth in KDM6A-null patient-derived cell line xenografts ⁷ and causes increased cell death in cell lines from muscle-invasive bladder cancers harboring KDM6A mutations ⁴². A phase I clinical trial combining the EZH2 inhibitor tazemetostat with immune checkpoint therapy in 12 patients including individuals with advanced urothelial carcinoma was published in 2019 and last updated in January 2023 (NCT03854474). The investigators reported partial response in 3 patients and stable disease in another 3 patients ⁴³. Although these tumors were not analyzed for KDM6A alterations, it is tempting to speculate that these responders included patients with KDM6A deficient cancers.

Conclusions

Our study identified KDM6A expression loss as a frequent molecular event in urothelial bladder cancer which preferentially occurs in low grade and non-invasive urothelial neoplasms. Truncating KDM6A mutations are the main but not the only cause for KDM6A expression loss. As KDM6A expression loss was not observed in non-neoplastic urothelium, we are now routinely applying KDM6A IHC to every newly diagnosed urothelial carcinoma and in case of flat urothelium with atypia of unknown significance. This procedure enables us to diagnose unequivocal dysplasia in a fraction of cases and to identify patients with KDM6A deficient tumors for which KDM6A IHC may be helpful for assessing subsequent biopsies or cytological specimens.

ACKNOWLEDGEMENTS

We are grateful to Laura Behm, Inge Brandt, Maren Eisenberg, Sünje Seekamp, Sina Dietrich, Tessa Sagau, Jana Hagemann, and Gabi Rieck for excellent technical assistance

CONFLICTS OF INTEREST STATEMENT

The mouse monoclonal antibody, clone HMV-314 was provided from ardoci GmbH (owned by a family member of GS)

FUNDING

None.

AUTHORS’ CONTRIBUTIONS

FV, NG, RS, GS, MK, SM: contributed to conception, design, data collection, data analysis and manuscript writing.

SB, AF, HP, SH, KF, SW, BR, AF, NB, BE, FR, SS, SE, ML, EB, AHM, HS, MF, MR, HZ, MS, KK, TE, SK, NA, JW, TK, TS, DH: participated in pathology data analysis, data interpretation, and collection of samples

FV, RS, MK, CHM: data analysis

FV, RS, GS, SM: study supervision

All authors agree to be accountable for the content of the work.

ETHICAL CONSIDERATION

The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

History

Received: September 30, 2024

Accepted: December 9, 2024

Figures and tables

Figure 1. KDM6A staining. The panels show strong nuclear staining of (a-b) the normal urothelium of the urinary bladder, (b) often with less intense staining of the umbrella cells (indicated by arrows in the inset) as compared to the remaining epithelial cells, loss of KDM6A staining in (c) a non-invasive pTa low grade, in (d) a non-invasive pTa high grade carcinoma, and in (e-f) two muscle invasive urothelial carcinomas, and retained KDM6A staining in (g) a non-invasive pTa and (h) a muscle invasive carcinoma.

Figure 2. Lack of prognostic relevance of KDM6A expression in a) all muscle invasive (pT2-4) urinary bladder cancers and in b) subsets of tumors grouped after presence of absence of KDM6A expression and Y-chromosome loss.

Figure 3. Examples of urothelial carcinomas with KDM6A expression loss but lack of truncating KDM6A mutations. a) pTa grade 2, female, KDM6A Glu1051Gln; b) pT2 grade 3, female, KDM6A wildtype; c-d) pT3 grade 3, female, KDM6A wildtype; e) pTa grade 2, male, KDM6A Gly1016Arg; f-h) pTa grade 2, male, KDM6A wildtype.

Figure 4. KDM6A immunostaining of conventional whole sections of a) a cancer with heterogeneous loss of KDM6A expression (overview) and b) magnification of the same tumor (see square in panel a). C) Tumor with focally reduced KDM6A staining. D) Tumor with strong KDM6A staining. All tumors harbored truncating KDM6A mutations.

Table I. Study cohort.

	Study cohort on TMA (n=2710)
Follow-up	636
Months
Mean	26.7
Median	15.0
Pathological tumor stage
pTa	887 (38.4%)
pT1	49 (2.1%)
pT2	462 (20.0%)
pT3	615 (26.6%)
pT4	298 (12.9%)
Tumor grade
G1	7 (0.3%)
G2	812 (30.3%)
G3	1858 (69.4%)
Pathological lymph node status
pN0	734 (62.0%)
pN+	449 (38.0%)
Resection margin status
R0	595 (80.6%)
R1	143 (19.4%)
Lymphatic vessel infiltration
L0	275 (49.5%)
L1	281 (50.5%)
Blood vessel infiltration
V0	450 (74.4%)
V1	155 (25.6%)
Percent in the column “study cohort on TMA” refers to the fraction of samples across each category. Numbers do not always add up to 2,710 in the different categories because of cases with missing data.

Table II. KDM6A expression and bladder cancer phenotype.

		KMD6A IHC result
	n	Loss (%)	Retained (%)	P
All cancers	2125	22.8	77.2
pTa G1	5	60.0	40.0
pTa G2 low	345	35.7	64.3	0.0004
pTa G2 high	152	23.0	77.0
pTa G3	92	18.5	81.5
pT2	378	17.2	82.8	0.1894
pT3	503	21.9	78.1
pT4	247	18.2	81.8
G2*	85	18.8	81.2	0.8466
G3*	1021	19.7	80.3
pN0*	546	18.9	81.1	0.7870
pN+*	378	19.6	80.4
R0*	463	21.2	78.8	0.4856
R1*	115	18.3	81.7
L0*	209	17.7	82.3	0.4138
L1*	231	20.8	79.2
V0*	355	18.3	81.7	0.1894
V1*	126	23.8	76.2
Pn0*	55	20.0	80.0	0.4703
Pn1*	79	25.3	74.7
Abbreviations: pT: pathological tumor stage, G: Grade, pN: pathological lymph node status, R: resection margin status, L: lymphatic invasion, V: venous invasion, PNI = perineural invasion; * pT2-4 cancers only

Table III. KDM6A expression, sex and Y chromosome loss.

				KDM6A IHC result
Parameter	Status	Subset	n	Loss (%)	Retained (%)	p
Patient gender	Female	pT2-4	338	15.4	84.6	0.0067
	Male		920	22.2	77.8
	Female	pTa	101	20.8	79.2	0.0239
	Male		478	31.8	68.2
Y-chromosome	Y loss	male, pT2-4	238	36.1	63.9	< 0.0001
	No loss		607	16.3	83.7
	Y loss	male, pTa	64	57.8	42.2	< 0.0001
	No loss		197	25.4	74.6

Table IV. KDM6A sequencing results.

Sex	Tumor stage	Grade	KDM6A IHC status	KDM6A mutation status	KDM6A mutation type	Biological significance
Female	pTa	G2	Loss	mut	Leu1340_Lys1345del	Truncating
Female	pTa	G3	Loss	mut	Ser876*	Truncating
Female	pTa	G2	Loss	mut	Lys685fs	Truncating
Female	pTa	G2	Loss	mut	Arg1127fs	Truncating
Female	pTa	G2	Loss	mut	Glu1051Gln	Unknown
Female	pT3	G3	Loss	mut	X1183_splice	Truncating
Female	pT2	G3	Loss	wt	wt
Female	pT3	G3	Loss	mut	Ser689fs	Truncating
Female	pT3	G3	Loss	mut	Glu206*	Truncating
Female	pT2	G3	Loss	mut	X1392_splice	Truncating
Female	pT2	G3	Loss	mut	Ala391fs	Truncating
Female	pT2	G3	Loss	mut	Phe289fs	Truncating
Female	pT2	G3	Loss	mut	Pro1191fs	Truncating
Female	pT1	G2	Loss	mut	Glu1217_Phe1219del	Truncating
Female	pT3	G3	Loss	wt	wt
Female	pT3	G3	Loss	mut	Gln329*	Truncating
Female	pT4	G3	Loss	mut	X129_splice	Truncating
Female	pT2	G3	Loss	mut	Pro1195fs	Truncating
Female	pT3	G3	Loss	mut	X1095_splice	Truncating
Female	pT3	G3	Loss	wt	wt
Female	pT3	G3	Loss	mut	p.Trp127*	Truncating
Female	pTa	G3	Retained	mut	Thr584Met	Unknown
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G3	Retained	wt	wt
Female	pTa	G2	Retained	mut	X189_splice	Truncating
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	mut	Asp1360fs	Truncating
Female	pTa	G2	Retained	mut	Gly359fs	Truncating
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	mut	X148_splice	Truncating
Female	pTa	G2	Retained	wt	wt
Female	pTa	G3	Retained	wt	wt
Female	pTa	G2	Retained	mut	Thr842fs	Truncating
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	wt	wt
Female	pTa	G2	Retained	mut	Gln805*	Truncating
Male	pTa	G2	Loss	mut	Tyr215fs	Truncating
Male	pTa	G2	Loss	mut	Gly1016Arg	Unknown
Male	pTa	G2	Loss	mut	Leu376fs	Truncating
Male	pTa	G2	Loss	mut	Gln1264*	Truncating
Male	pTa	G2	Loss	mut	Gln607*	Truncating
Male	pTa	G2	Loss	wt	wt
Male	pTa	G2	Loss	mut	X944_splice	Truncating
Male	pTa	G2	Loss	mut	Gln369*	Truncating
Male	pTa	G2	Loss	wt	wt
Male	pTa	G2	Loss	wt	wt
Male	pTa	G2	Loss	mut	Leu617fs	Truncating
Male	pTa	G2	Loss	mut	X112_splice	Truncating
Male	pTa	G2	Loss	mut	Tyr228fs	Truncating
Male	pTa	G2	Loss	mut	Asn604fs	Truncating
Male	pTa	G2	Loss	mut	X189_splice	Truncating
Male	pTa	G2	Loss	mut	Ser1068*	Truncating
Male	pTa	G2	Loss	mut	Leu956*	Truncating
Male	pTa	G2	Loss	mut	Leu617fs	Truncating
Male	pTa	G2	Loss	wt	wt
Male	pTa	G2	Loss	mut	Leu956*	Truncating
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	mut	Val1241del	Unknown
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt
Male	pTa	G2	Retained	wt	wt