TYMS overexpression is frequent and linked to grade progression in urothelial bladder cancer

doi:10.21203/rs.3.rs-4898911/v1

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TYMS overexpression is frequent and linked to grade progression in urothelial bladder cancer

https://doi.org/10.21203/rs.3.rs-4898911/v1

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Purpose:

While elevated Thymidylate synthase (TYMS) levels are observed in many cancers, its specific role in bladder cancer remains unclear. This study aims to clarify its activity in a large tumor cohort.

Methods:

A tissue microarray (TMA) containing over 2,700 bladder tumors was analyzed using immunohistochemistry and fluorescence in-situ hybridization (FISH) to assess TYMS expression and gene amplification.

Results:

TMYS immunostaining was detectable in 83.1% of 1,799 analyzable bladder cancers. The fraction of cancers with moderate to strong TMYS positivity increased markedly from pTaG2 low (6.5%) to pTaG2 high grade (20.7%), and pTaG3 cancers (29.0%; p < 0.0001). There was also a significant increase of moderate to strong staining from pTa to advanced stage pT2-4 cancers (pTa 13.2% vs. pT2-4 32.9% p < 0.0001). In muscle-invasive cancers, the frequency of TMYS immunostaining increased with tumor grade (p = 0.0007), but there was no association between TYMS expression and patient prognosis (p = 0.4365). TYMS amplification was found in 3.1% of 1,775 analyzable bladder cancers. TYMS amplification increased from pTa (0.6%) to pT2-4 (3.7%; p < 0.0001), but in muscle invasive-cancers TYMS copy number alterations were unrelated to tumor phenotype and patient prognosis. Strong TYMS positivity was significantly associated with TYMS amplification (p = 0.0096) but only a subset (28.6%) of amplified cancers showed a strong TMYS staining and only 7.2% of cancers with strong TYMS expression had a TYMS amplification.

Conclusion:

TYMS overexpression plays a role in early bladder cancer development and grade progression, but its expression is largely unrelated to the disease course in muscle-invasive cancers. Gene amplification is not the primary driver of TYMS protein overexpression.

Bladder cancer belongs to the ten most common cancer types worldwide [1]. Around 80% of patients initially present with either non-invasive (pTa) or early invasive (pT1) cancers, which generally have a more favorable prognosis and can mostly be addressed through bladder-sparing approaches. However, nearly all of these tumors recur, and approximately 20% of pT1 carcinomas progress to muscle-invasive disease [2–4]. For those with muscle-invasive bladder carcinomas, treatment typically involves radical cystectomy or radiotherapy both combined with systemic therapies [5]. Despite these interventions, almost 50% of patients with pT2-4 cancers experience early metastasis and eventual die from the disease. A better understanding of the molecular features driving urothelial carcinoma will ultimately lead to improved individual patient prognostication and optimized treatment decisions.

The Thymidylate synthase (TYMS) is an enzyme which is involved in the synthesis of thymidine monophosphate (dTMP) by the reductive methylation of deoxyuridine monophosphate (dUMP). This reaction is a critical step in the thymidine biosynthesis pathway, ensuring a supply of one of the four DNA nucleotides required for DNA replication and repair [6]. Several lines of evidence suggest a role of deregulated TYMS expression in cancer [7]. For example, studies in gastric, colon or prostate cancer have demonstrated that TYMS activity is higher in cancerous than in normal tissues and also correlated with cancer progression [8–10]. Of particular interest, TYMS is a target for cancer chemotherapeutic agents such as 5-Flourouracil (5-FU) [10–15], Methotrexat [16], or Permetrexed [17]. Several studies have suggested that high levels of TYMS expression in tumor cells may be associated with resistance to TYMS inhibitors, while low TYMS expression may predict a better response to treatment [11–15, 17, 18]. Studies on 54–176 urothelial carcinomas demonstrated a significant upregulation of TYMS in a significant fraction of cases [18–20]. The TYMS gene is located at 18p11.32, a region which has been shown to carry amplifications in urothelial carcinoma in previous studies [21–24]. TYMS amplification could potentially lead to elevated levels of TYMS expression.

To learn more on the prevalence and clinical significance of TYMS expression in bladder cancer and the role of gene amplification for TYMS upregulation, 2,710 urothelial carcinomas of the urinary bladder were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in a tissue microarray (TMA) format.

Tissue Microarrays (TMA). The TMAs used in this study were first employed in a study on the prognostic role of GATA3 expression in urothelial bladder carcinomas [25]. 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, Institute of Pathology, Charité Berlin, Department of Pathology, Academic Hospital Fuerth, or Department of Pathology, Helios Hospital Bad Saarow, and/or treated at Department of Urology, University Hospital Hamburg, Department of Urology, Charité Berlin, Department of Urology, Helios Hospital Bad Saarow, Department of Urology, Albertinen Hospital Hamburg (all in Germany), and Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland between 2005 and 2021. Patients at each center were treated according to the guidelines at the time. In brief, patients with pTa disease underwent a transurethral resection of the bladder tumor with or without postoperative instillation therapy, while 1,826 patients with pT2-pT4 disease were treated by mostly by radical cystectomy. Available histopathological data including grade, tumor stage (pT), lymph node status (pN), and status of venous (V) and lymphatic (L) invasion are shown in Table 1. Clinical follow up data were available from 626 patients with pT2-4 carcinomas treated by cystectomy. The overall survival time of these patients ranged from 1 to 176 months (median: 26.7 months, median: 15 months). Tissues were fixed in 4% buffered formalin and then embedded in paraffin. The TMA manufacturing process has previously been described in detail [25]. In brief, one tissue spot (diameter: 0.6 mm) per patient was taken from a tumor containing tissue block. 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 analysis had been approved by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

Immunohistochemistry. Freshly cut TMA sections were immunostained on one day and 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 Tris-EDTA-Citrat (TEC) puffer. Endogenous peroxidase activity was blocked with Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Santa Clara, CA, USA; #S2023) for 10 minutes. Primary antibody specific for TYMS protein (recombinant rabbit monoclonal, HMV-305, MS Validated Antibodies, Hamburg, Germany; #6504 − 305) was applied at 37°C for 60 minutes at a dilution of 1:150). Bound antibody was then visualized using the Dako REAL EnVision Detection System Peroxidase/DAB+, Rabbit/Mouse kit (Agilent Technologies, Santa Clara, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with hemalaun. For tumor tissues, the percentage of TYMS positive neoplastic cells was estimated, and the staining intensity was semi-quantitatively recorded (0, 1+, 2+, 3+). For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered negative. Tumors with 1 + staining intensity in ≤ 70% of tumor cells and 2 + intensity in ≤ 30% of tumor cells were considered weakly positive. Tumors with 1 + staining intensity in > 70% of tumor cells, 2 + intensity in 31–70%, or 3 + intensity in ≤ 30% of tumor cells were considered moderately positive. Tumors with 2 + intensity in > 70% or 3 + intensity in > 30% of tumor cells were considered strongly positive.

Fluorescence in-situ hybridization (FISH). Five micrometer TMA sections were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced denaturation for 10 minutes in a water bath at 99°C in P1 pretreatment solution (Agilent Technologies, Santa Clara, CA, USA; #K5799). For proteolytic treatment, slides were added to VP2000 protease buffer (Abbott, North Chicago, IL, USA; #2J.0730) for 200 minutes at 37°C in a water bath. A commercial FISH probe kit containing both, gene specific 18p11.32 and corresponding centromere probes were utilized for copy number detection of TYMS (TYMS gene locus and centromere 18; self-designed, Empire Genomics, Buffalo, NY, USA). Hybridization was performed overnight at 37°C in a humidified chamber. Posthybridization washes was done according to the manufacturer’s direction (Agilent Technologies, Santa Clara, CA, USA; #K5799). Nuclei were counterstained with 125 ng/ml 4’,6-diamino-2-phenylindole in antifade solution (Biozol; Eching, Germany; #VEC-H-1200). Stained tissues were manually interpreted with an epifluorescence microscope and copy numbers of 18p11 and centromere 18 were estimated for each tissue spot as previously described [26]. A tumor was considered amplified if predominant tumor cell population had at least twice as many 18p11 signals than centromere 18 signals (ratio ≥ 2.0). A detailed scoring of 20 cells per sample was executed in cases with equivocal results (i.e. relative increase of 18p11 signals but absence of clearcut amplification). Cancers with equal signal numbers of 18p11 and centromere 18 were considered as non-amplified. Presence of fewer 18p11 signals than centromere 18 signals in at least 60% of all tumor nuclei in a tumor spot was considered as heterozygous deletion. Absence of 18p11 signals in the presence of centromere 18 signals in all tumor nuclei and presence of 18p11 signals in normal cell nuclei was considered as homozygous deletion [27]. All other tumors were considered non-deleted.

Statistics. Statistical calculations were performed with JMP17® software (SAS®, Cary, NC, USA). Contingency tables and the chi²-test were performed to search for associations between TYMS immunostaining, TYMS copy number status, and tumor phenotype. Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant differences between groups.

Technical issues. Of our 2,710 urothelial carcinomas, 1,799 (66.4%) were interpretable for TYMS IHC and 1,775 (65.5%) for FISH. Non-informative cases were caused by a lack of unequivocal tumor cells on the TMA spots or absence of entire tissue spots on the TMA.

TYMS immunostaining in urothelial carcinomas. TYMS staining in normal urothelium was usually completely absent and occasionally weak and focal. TYMS positivity in tumors was dominantly cytoplasmic but nuclear staining also occurred. It was observed in 83.1% of all urothelial carcinomas including 56.4% with weak, 14.4% with moderate, and 12.3% with strong staining. Representative images of TYMS immunostaining are shown in Fig. 1. The relationship between TYMS staining and tumor phenotype is given in Table 2. Within pTa tumors moderate to strong TMYS positivity increased markedly from pTaG2 low (6.5%) to pTaG2 high (20.7%), and pTaG3 cancers (29.0%; p < 0.0001). There was a further increase of moderate to strong positivity from pTa (13.5%) to pT2-4 cancers (32.9%; pTa vs. pT2-4, p < 0.0001). Within muscle-invasive carcinomas, the TYMS expression level was higher in G3 (88.5%) than in G2 tumors (75.5%; p = 0.0007) while there was no clear-cut relationship with other parameters of cancer aggressiveness. The statistically significant relationship between TYMS expression and pT status (p = 0.0011) was driven by a higher fraction of strongly positive cases in pT3 than in pT2 or pT4 cancers. In 412 patients with pT2-4 carcinomas that were treated by cystectomy, TYMS staining was unrelated to overall survival (p = 0.4365, Fig. 2A).

TYMS copy number status in urothelial bladder carcinomas. TYMS amplification occurred in 55 of 1,775 successfully analyzed cancers (3.1%). The TYMS amplification rate increased from pTa (0.6%) to pT2-4 (3.7%; p < 0.0001; Table 2). TYMS deletions were rare and only detected in 16 (1.4%) of 1,115 pT2-4 carcinomas. In pT2-4 carcinomas, TYMS alterations were unrelated to parameters of the tumor phenotype and patient prognosis (Table 2, Fig. 2B). Examples of FISH findings were shown in Fig. 3. TYMS immunostaining was significantly associated with TYMS copy number status. (p = 0.0096). However, only 28.6% of 42 amplified cancers showed strong TMYS staining and only 7.2% of strongly TYMS positive cancers had a TYMS amplification (Fig. 4).

Our study findings demonstrate that TYMS overexpression is frequent in urothelial neoplasms and strongly related to poor histologic grade, especially in non-invasive cancers. However, 18p11 amplification is not the major mechanism for TMYS upregulation.

Our data illustrates that a marked TYMS overexpression occurs in a significant fraction of urothelial carcinomas. Infrequent (15%) with low TYMS expression in normal urothelium and a more frequent (70%) with higher expression in urothelial carcinomas has also been described by Li et al. [18]. Our TYMS positivity rate of 83.1% (56.4% weak, 14.4% moderate, 12.3% strong) is slightly higher than in previous studies which reported TYMS positivity in 70% of 67 [18], 44% of 176 [20], and 48% of 54 patients [19]. Variations of the positivity rate between our data and the previous three studies may be caused by different staining protocols and antibodies, criteria for defining positivity, and the composition of the patient cohorts. For example, Li et al. [18] and Ide et al. [20] defined tumors as positive if > 25% of tumor cells showed a moderate or strong TYMS staining. Of note, our TYMS IHC assay was previously validated according to the guidelines of the International Working Group for Antibody Validation (IWGAV) [28] by comparison with RNA data and a comparison with a second antibody on 76 different normal tissue types (manuscript submitted). Such an extensive normal tissue validation exposes antibodies to most proteins occurring in adult humans and thus enables the identification of most antibody cross-reactivities.

In our cohort of 1,799 analyzable urothelial bladder carcinomas, a significant increase of moderate and strong TYMS positivity was seen with grade progression in pTa tumors and also within pT2-4 cancers while the TYMS expression level was not unequivocally related to other parameters of aggressive disease within 870 muscle-invasive carcinomas. These observations are in agreement with findings of Li et al. who found an association of TYMS positivity with grade in pTa-T1 urothelial cancer [18]. The high rate of TYMS positivity even in low grade pTa tumors as compared to the TYMS negativity of normal tissues, the significant increase of TYMS expression with grade in pTa tumors, and the lack of a striking further increase of TYMS expression with stage progression and metastasis argues for a significant role of TYMS overexpression in the early phase of developing urothelial neoplasms preceding the progression to invasive disease. It is of note, that other authors had described significant association between high TYMS expression and vascular invasion in a cohort of 54 bladder cancer [19], and lymphovascular invasion or nodal metastasis in a series of 176 cancers [20].

The low rate of TYMS amplification (3.1%) found in our patients is consistent with data from two earlier studies describing only rare TYMS amplification in bladder cancer patients [22, 23]. Although there was a significant association between TYMS amplification and overexpression our data demonstrate that amplification is neither required nor sufficient to induce detectable TYMS expression. That 7.1% of the 42 TYMS amplified cases did not show any detectable TYMS expression is consistent with earlier data from the analysis of other amplified genomic areas showing that only proteins that are already expressed can be overexpressed in case of an amplification of its gene [29–31]. That only 7.2% of the cancers with strong TYMS immunostaining were amplified demonstrates that gene copy number changes are not the mayor cause for increased TYMS expression. Other known causes for TYMS overexpression include epigenetic regulations of the chromatin structure [32, 33], polymorphisms of TYMS promoter [34, 35] or overexpression of the TYMS associated transcription factor E2F1 [36].

The mechanism by which elevated TYMS expression contributes to cancer development and/or progression is not fully understood. TYMS is a key enzyme in the de novo synthesis of thymidine monophosphate (dTMP), which is essential for DNA synthesis and repair. Thus, high levels of TYMS can lead to increased DNA synthesis in rapidly dividing cells, supporting their growth and proliferation - an important factor leading to the development of cancer. This aligns with positive association of high TYMS levels to proliferation markers like Ki67 [19, 37–39]. In-vitro knockdown of the TYMS gene suppressed proliferation and enhanced apoptosis in various cancer cell lines [39–41]. Moreover, TYMS can also regulate relevant genes in cancer development. In lung cancer, TYMS has been shown to contribute to epithelial-to-mesenchymal transition (EMT) by upregulating HOXC6 and HMGA2 and downregulating AXL, SPARC, and FOSL1 [42]. EMT, a hallmark of cancer, is pivotal for cancer progression and allows solid tumors to become more malignant by increasing invasiveness and metastatic activity.

Consequently it is not surprising that studies in many cancer types, like colorectal [9, 12, 43], gastric [10, 13], lung [44], breast [39] and prostate cancer [8] have demonstrated a dismal prognostic role of TYMS overexpression. The lack of a prognostic role of TYMS expression in bladder cancer is in line with the notorious difficulty in finding molecular prognostic parameters for application in muscle-invasive urothelial carcinoma. Various other molecular alterations such as, p53 alterations [45, 46], HER2 [47, 48], and c-Myc [49], which are strongly linked to prognosis in many other tumors have failed to demonstrate unequivocal prognostic utility in bladder cancer. Even histological grading does not have a confirmed prognostic role in pT2-4 urothelial cancers, leading to the ongoing WHO recommendation not to grade these advanced-stage tumors since 2004 [50].

TYMS expression analysis may also have a predictive role as TYMS is a major target for antifolate therapies, especially 5-FU [14]. TYMS amplification [15, 17, 51, 52] and variations of its protein expression [10–14] correlated with the response to therapy in various cancers. Overall, the data suggest that elevated TYMS levels are generally linked to resistance against TYMS inhibitors, whereas lower TYMS expression tends to correlate with a more favorable treatment response. Data on the predictive role of TYMS levels in urothelial cancer is limited. Li et al. reported a more favorable response to 5-FU-based adjuvant therapy in TYMS positive than in TYMS negative pTa-1 tumors in a study on 67 patients [18]. In another study on 52 patients undergoing nephroureterectomy for invasive urothelial cancer, TYMS expression had no measurable effect on adjuvant 5-FU therapy outcomes [53]. Despite the decreasing use of 5-FU in non-muscle invasive bladder and upper urinary tract cancers, it remains an integral component of chemoradiation in muscle invasive cancer [54, 55]. Predicting biomarker are still missing in this bladder preserving strategy. Based on observations in other cancer types, it might be worthy to perform further studies on the predictive role of TYMS expression levels for the efficacy of 5-FU based therapies in bladder cancer patients undergoing chemoradiation.

In conclusion, our data demonstrate a role of TYMS overexpression in early bladder cancer development and grade progression but also show that TYMS expression levels are largely unrelated to the disease course in muscle-invasive cancers.

EMT: epithelial-to-mesenchymal transition

5-FU: 5-Flourouracil

FISH: fluorescence in-situ hybridization

IHC: immunohistochemistry

IWGAV: international working group for antibody validation

TMA: tissue microarray

TYMS: thymidylate synthase

Acknowledgements

We are grateful to Melanie Steurer, Laura Behm, Inge Brandt, Sünje Seekamp, Sascha Eghtessadi, Silvia Schnöger, Heike Jordan, Vivian Modi, and Lucy Joan Veloz Ramirez for excellent technical assistance.

Supported by a Ferdinand Eisenberger grant of the Deutsche Gesellschaft für Urologie (German Society of Urology), grant ID PlH1/FE-23.

Conflict of interest

The antibody clone HMV-422 were obtained from MS Validated Antibodies GmbH (owned by a family member of GS).

Authors Contribution Statement

HP, MK, RS, TS, JW, GS: contributed to conception, design, data collection, data analysis and manuscript writing.

HP, ML, SM, FR, SS, SE, AF, AHM, HS, SK, DH: participated in pathology data analysis and data interpretation.

SH, KF, SW, FR, SS, SE, AF, AHM, HS, MF, MR, TE, SK, TK, DH, HZ, MS, KK: collection of samples.

HP, RS, MK: data analysis.

HP, MK, TS, RS, GS: study supervision.

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

Funding

No funding

Ethics approval

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.

Data availability statement

All data generated or analyzed during this study are included in this published article.

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04 Oct, 2024
Reviews received at journal
29 Sep, 2024
Reviewers agreed at journal
24 Sep, 2024
Reviewers agreed at journal
24 Sep, 2024
Reviewers invited by journal
24 Sep, 2024
Editor assigned by journal
15 Aug, 2024
Submission checks completed at journal
14 Aug, 2024
First submitted to journal
12 Aug, 2024

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TYMS overexpression is frequent and linked to grade progression in urothelial bladder cancer

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Version 1