Prostate cancer survivors with symptoms of radiation cystitis have elevated fibrotic and vascular proteins in urine.

Radiation for pelvic cancers can result in severe bladder damage and radiation cystitis (RC), which is characterized by chronic inflammation, fibrosis, and vascular damage. RC development is poorly understood because bladder biopsies are difficult to obtain. The goal of this study is to gain understanding of molecular changes that drive radiation-induced cystitis in cancer survivors using urine samples from prostate cancer survivors with history of radiation therapy. 94 urine samples were collected from prostate cancer survivors with (n = 85) and without (n = 9) history of radiation therapy. 15 patients with radiation history were officially diagnosed with radiation cystitis. Levels of 47 different proteins were measured using Multiplex Luminex. Comparisons were made between non-irradiated and irradiated samples, and within irradiated samples based on radiation cystitis diagnosis, symptom scores or hematuria. Statistical analysis was performed using Welch's t-test. In prostate cancer survivors with history of radiation therapy, elevated levels of PAI 1, TIMP1, TIMP2, HGF and VEGF-A were detected in patients that received a radiation cystitis diagnosis. These proteins were also increased in patients suffering from hematuria or high symptom scores. No inflammatory proteins were detected in the urine, except in patients with gross hematuria and end stage radiation cystitis. Active fibrosis and vascular distress is detectable in the urine through elevated levels of associated proteins. Inflammation is only detected in urine of patients with end-stage radiation cystitis disease. These results suggest that fibrosis and vascular damage drive the development of radiation cystitis and could lead to the development of more targeted treatments.

Introduction

Approximately 3.6 million prostate cancer (PCa) survivors are currently living in the United States, representing 1/5th of all cancer survivors [1]. It is estimated that, in the United States, 191,390 new patients will receive a PCa diagnosis in 2020. Most of these PCa are diagnosed at an early stage, which contributes to a very high overall 5-year survival rate (99%) [2]. Treatment of PCa is dependent on stage at diagnosis, risk of reoccurrence, patient’s age, presence of comorbidities and patient’s personal preference, and includes active surveillance, surgery, and radiation, chemotherapy, hormone, and androgen-deprivation therapies [1]. Despite the high survival rate after PCa, many PCa survivors struggle with short or long-term side effects from their cancer therapy. Approximately half the PCa survivors that received radiation therapy or surgery suffer from urinary or bowel dysfunction [1]. Of the PCa survivors that were treated with radiation therapy, a small percentage (range 4–11%) will develop radiation (hemorrhagic) cystitis (RC) that increases with years after radiation therapy [3].

RC is a chronic bladder condition that is characterized by urinary frequency, nocturia, incontinence, pelvic pain and hematuria. These symptoms are caused by severe bladder fibrosis and vascular damage for which no cure exists to date. Treatments are generally focused on arresting bleeding and include blood transfusions, cystoscopy with fulguration/clot evacuation, hyperbaric oxygen therapy, instillation of aminocaproic acid, alum, silver nitrate or formalin, and cystectomy with or without neobladder reconstruction [3-5]. RC treatments have limited effectiveness, are time consuming and can have serious side effects. Thus, there is a dire need for improved treatment options for patients suffering from RC.

In addition to the lack of safe and effective treatments, RC is often diagnosed at a late stage when damage may be irreversible, making it more challenging to effectively treat. Lack of mechanistic insight into the disease progression is in part due to lack of bladder biopsies available for analysis; taking bladder biopsies from RC patients is risky as it may cause additional damage to an already fragile tissue. However, insight into molecular changes after irradiation and RC diagnosis is essential to identify patients early on that are at increased risk for developing RC and to develop targeted treatments. To circumvent the inability to obtain tissue biopsies, we used urine samples of PCa survivors as ‘liquid biopsies’ to identify altered protein levels. In urological research, urinary biomarkers are widely considered for carcinoma in situ, bladder cancer and interstitial cystitis, but are novel for RC [6, 7].

RC bladders are believed to have three main histological characteristics: accumulation of tissue fibrosis, vascular damage as evidenced by hematuria, and chronic inflammation. Therefore, in this study, we used urine samples from PCa survivors with a history of radiation therapy to identify changes in excreted urinary proteins involved in fibrosis, inflammation and vascular biology.

Materials and methods

Urine and data collection

The study was conducted with full Beaumont Institutional Review Board (IRB) approval (#2015–302 and #2018–179). Study participants were recruited at Hospital Clínico San Borja Arriarán, Santiago de Chile, Chile, with approval of the Chilean Comité Ético-Científico (CEC-SSMC # 96/16). Patient population included in the study are Chilean PCa survivors with and without history of external beam radiation therapy. Prostate cancer survivors that concurred to regular control of their prostate cancer, were invited to participate in the study between March 2016 and March 2020. Of those that received radiation treatment, some patients had been diagnosed with RC. RC diagnosis was determined independently by a urologist through cystoscopy, and defined by a pale bladder mucosa and telangiectasia, with or without ulcers, and a decreased maximal bladder capacity as measured during cystoscopy [8]. Exclusion criteria for participation were a history of chemotherapy, interstitial cystitis, recurrent urinary tract infections, kidney and/or bladder stones, prostatitis, or other bladder disorders (e.g. neurogenic bladder) [8]. Participants voluntarily signed a written informed consent before filling out a health survey and providing a midstream urine sample. The survey included questions on demographics, smoking status, bladder health, symptom severity, and radiation history. For symptom severity, patients scored the incidence of nocturia, incontinence, hematuria, gross hematuria, urgency, and urogenital spasm on a scale from zero (never) to five (multiple times per day) [9]. Scores for each symptom were added to form the total score (maximum = 30). High symptom score was defined as score ≥ 10. All patients with micro- and macrohematuria were studied with cystoscopy to confirm RC and to rule out other causes of hematuria. Urine was collected in sterile urine cups at a random time point (e.g. not first urine of the day) and urine preservative (Norgen Biotek) was immediately added to keep urinary proteins stable for up to one year at room temperature. De-identified urine samples and surveys were shipped to William Beaumont Hospital’s Research Institute, Royal Oak, MI. Dipstick analysis was performed on urine samples to rule out urinary tract infections and identify presence of hematuria. Urine samples were spun down for 5 minutes at 700 x g and supernatant was stored in aliquots at -80°C.

Multiplex luminex assay

Urine samples were thawed on ice and vortexed prior to use. Only urine samples not previously thawed were used for this study. Fibrotic, inflammatory and vascular proteins (Table 1) were measured using the Milliplex multiplex assay system (EMD Millipore) according to the manufacturer’s instructions. Samples were run in duplicate simultaneously with known standards and quality control samples on Bio-Plex 200 System (Bio-Rad). This system was previously used to for development or urine biomarkers for interstitial cystitis [6]. Groups that were compared included non-irradiated versus irradiated samples. Of the samples that received radiation therapy, protein changes in the urine were analyzed based on the presence of RC diagnosis, hematuria, and high symptom score.

Table 1

Detectability of fibrotic, inflammatory and angiogenic proteins in urine samples of prostate cancer survivors using Luminex assay.

	Fully detectable			Partially detectable			Minimally detectable
	Proteins	LLD	Avg	Proteins	LLD	Avg	Proteins	LLD	Avg
Fibrotic factors	Cathepsin-D TIMP-1 TIMP-2	24 20 49	55356 321 1546	MMP-9 MMP-10 MMP-12 PAI-1 TIMP-3 TIMP-4	14.0 27.0 98.0 2.0 98.0 10.0	783 15 32 9.4 31 5.7	MMP-1 MMP-2 MMP-3 MMP-7 MMP-13 TGFb1	27 68 146 548 58 9.8	07.20000.5
Inflammatory factors	MCP-1 MCSF sVCAM-1	3.2 97.6 61	572 51221 1129	sICAM-1	24.0	256	Fractalkine GMCSFIL-1a*IL-1bIL-4IL-6*IL-7IL-8IL-10IL-13IL-17aMIP-1a* TNFa	3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2 3.2	0 0.4 0.8 0.02 1.1 2.1 0 20.9 0.1 0.06 0.01 0.62 0.63
Vascular factors	EGFHB-EGFHGFPlGFVEGF-A	2.7 1.4 27.4 1.4 13.7	1092 16 113 52 168	Endoglin FGF-2 Leptin PDGF-AA	27.4 13.7 137 2.0	28 2 179 13	Angiopoetin-2BMP9 GCSF Follistatin PDGF-AB/BB VEGF-D	13.7 2.7 13.7 27.4 24 6.9	0.9 0.4 0.2 1.4 8.8 0.9

* Only detectable in 1–2 RC cases with gross hematuria; LLD = lower limit of detection (pg/ml); Avg = average detected protein level in samples (pg/ml).

Statistical analysis

Statistical analysis was performed using SPSS 26.0. A one-way ANOVA test followed by a post-hoc two-tailed Welch t-test was performed to determine statistical significance with unequal variance between two populations: non-irradiated versus irradiated groups (Fig 1), or, within irradiated group, patients with versus without RC diagnosis, hematuria or high symptom scores (Figs 2–4). Significance level was set at p ≤ 0.05. Results are expressed as mean ± standard deviation (SD).

Fig 1

Fibrotic and angiogenic proteins are not altered in urine after radiation therapy.

(A-C) The fibrotic proteins PAI 1, TIMP1, and TIMP2, and (E) the vascular protein VEGF-A are unaltered between non-irradiated (non-IRR) and irradiated (IRR) PCa survivors. (D) HGF levels are overall higher after irradiation treatment, though this is not significant. Non-IRR: n = 9; IRR: n = 85. Black line = mean; Error bar = SD.

Fig 2

PAI 1 and TIMP1/2 are altered in urine of prostate cancer survivors.

Increased levels of (A-C) PAI 1, (D-F) TIMP1 and (G-I) TIMP2 levels are measured in PCa survivors with history of radiation treatment with RC diagnosis, hematuria or high symptom score. No: n = 67; Yes: n = 15. Black line = mean; Error bar = SD.

Fig 4

Urinary inflammatory markers not altered in prostate cancer survivors with RC.

In prostate cancer survivors with radiation history, Inflammatory cytokines were not altered in patients with RC in comparison to patients without RC diagnosis. Y-axis: protein concentration in pg/ml. No: n = 70; Yes: n = 15. Black line = mean; Error bar = SD.

Results

Patient demographic

The patient population consists of PCa survivors with (n = 79) and without (n = 9) a history of radiation therapy. Of those that received radiation therapy, 15 patients were diagnosed with RC prior to urine collection. All urine samples were collected between March 2016 and March 2019. Patient demographic data, radiation history and symptom severity are summarized in Table 2. All patient groups were comparable in age, BMI and smoking status. Of the PCa survivors with radiation history, urine samples were collected on average 5.5 years after radiation exposure (4.07 yr for RC patients, and 5.8 yr for non-RC patients). The majority of patients received 36–40 radiation treatments, though dose did not appear to determine a patient’s risk for developing RC. Overall, more patients with RC suffered from frequency, urgency, nocturia, hematuria, urinary incontinence and bladder spasm. One non-RC case reported hematuria; this patient was diagnosed with RC after data collection.

Table 2

Patient demographics, radiation history and symptom severity.

		Participants with radiation treatment history
	Control	All	RC diagnosis
Total	(n = 9)	(n = 85)	Yes (n = 15)	No (n = 70)
Age (y)*	73.33 (± 9.72)	72.67 (± 7.39)	72.94 (± 9.69)	72.94 (± 6.85)
BMI (kg/m2)*	26.40 (± 4.36)	27.19 (± 3.30)	27.29 (± 3.04)	27.17 (± 3.37)
Smoking Status	n = 9	n = 83	n = 13	n = 70
Never	66.7% (n = 6)	44.6% (n = 37)	38.5% (n = 5)	45.7% (n = 32)
Past	33.3% (n = 3)	41.0% (n = 34)	53.8% (n = 7)	38.6% (n = 27)
Yes	0	14.4% (n = 12)	7.7% (n = 1)	15.7% (n = 11)
Diabetes	11.1% (n = 1)	21.7% (n = 18)	7.1% (n = 1)	24.6% (n = 17)
Years Post IRR*	n.a.	5.5 (±4.93)	4.07 (±2.70)	5.8 (±5.24)
Total Radiation dose (Gy)	n.a.	n = 85	n = 15	n = 70
< 36		7.1% (n = 6)	6.7%(n = 1)	7.1% (n = 5)
45–54		1.2% (n = 1)	0%	1.4% (n = 1)
55–63		7.1% (n = 6)	20% (n = 3)	4.3% (n = 3)
64–72		81.2% (n = 69)	73.3% (n = 11)	82.9% (n = 58)
73–81		3.5% (n = 3)	0%	4.3% (n = 3)
Symptom severity
Frequency (>8 voids)	28.6% (n = 2/7)	34.2% (n = 27/79)	57.1% (n = 8/14)	29.2% (n = 19/65)
9–11 voids	50% (n = 1)	66.7% (n = 18)	50% (n = 4)	73.7% (n = 14)
12–14 voids	50% (n = 1)	22.2% (n = 6)	25% (n = 2)	21.1% (n = 4)
> 14 voids	0%	11.1% (n = 3)	25% (n = 2)	5.3% (n = 1)
Urgency	0%	35.3% (n = 30/85)	53.3% (n = 8/15)	31.4% (n = 22/70)
Hematuria	0%	11.8% (n = 10/85)	60% (n = 9/15)	1.4% (n = 1/70)
Gross hematuria		50% (n = 5)	55.6% (n = 5)	0%
Nocturia	66.7% (n = 6/9)	78.8% (n = 67/85)	100% (n = 15/15)	74.3% (n = 52/70)
Once/night	16.7% (n = 1)	17.9% (n = 12)	20% (n = 3)	17.3% (n = 9)
Multiple times/night	83.3% (n = 5)	82.1% (n = 55)	80% (n = 12)	82.7% (n = 43)
Urinary incontinence	0%	9.4% (n = 8/85)	20% (n = 3/15)	7.1% (n = 5/70)
Bladder spasm	0%	5.9% (n = 5/85)	20% (n = 3/15)	2.9% (n = 2/70)

* Averages are given (± SD). BMI: body mass index; IRR: irradiation.

Altered urinary protein profile associated with symptoms of radiation damage

Multiplex Luminex assays were used to measure fibrotic, vascular and inflammatory proteins in human urine samples. Of the proteins tested, 22 proteins were within the detectable range of the Luminex assay in all or at least half (partially detectable) of the urine samples (Table 1).

Proteins in the urine were not altered in response to irradiation (Fig 1); only HGF levels, were increased in patients with radiation history, though this did not reach the 5% significance level (Fig 1D).

Within the PCa survivors with a history of radiation therapy, several fibrotic and vascular proteins were altered based on the presence of RC-related symptoms (RC diagnosis, hematuria or high symptom score). The fibrotic proteins PAI 1, TIMP1 and TIMP2 were detectable in the urine and their elevated levels were associated with RC diagnosis, hematuria or high symptom score (Fig 2). For PAI 1, its highest increase was seen in samples with hematuria; the three samples with the highest PAI 1 levels were patients with gross hematuria (Fig 2B). TIMP1 and TIMP2 were also elevated in PCa survivors with radiation history and RC symptoms (Fig 2D–2I). TIMP2 levels had a stronger association with high symptom score (Fig 2I), while TIMP1 levels were more closely associated with RC diagnosis (Fig 2D).

The vascular proteins HGF and VEGF-A were also altered in PCa with radiation history based on RC symptoms (Fig 3). HGF protein concentration was significantly higher in patients with high symptom scores and showed a positive association with hematuria and RC diagnosis (Fig 3A–3C). VEGF levels were significantly higher in patients with hematuria versus those that did not report hematuria (Fig 3E). The average VEGF levels were higher for patients with RC diagnosis and patients with high symptom scores, though these lacked statistical significance (Fig 3D–3F).

Fig 3

HGF and VEGF-A levels coincide with the presence of RC symptoms.

Levels of (A-C) HGF and (D-F) VEGF-A are elevated in PCa survivors with RC diagnosis, with hematuria or with a high symptom scores. No: n = 70; Yes: n = 15. Black line = mean; Error bar = SD.

Of the inflammatory proteins measured, only MCP-1, MCSF and sVCAM-1 were detectable in the urine. However, no association was found between their concentration and RC diagnosis, hematuria or symptom score (Fig 4). IL-1a, IL-6, and MIP-1a were detectable only in 1–2 severe RC cases that reported daily occurrence of gross hematuria. IL-8 was present in 11/94 urine samples, but was not associated with any RC or radiation related symptoms (Fig 4).

Discussion

RC is characterized in part by fibrosis, hematuria and inflammation. Lack of available bladder biopsies from patients with radiation history makes it challenging to identify pathways that drive the development of RC. As such, targeted therapies cannot be developed, and patients are left with suboptimal treatment options. Thus, we used urine samples as ‘liquid bladder biopsies’ to look for protein changes in PCa survivors with a history of radiation therapy. We previously identified three elevated vascular markers in the urine of PCa survivors with a history of radiation therapy [8]. We expanded this cohort and looked for changes in inflammatory and fibrotic proteins in addition to vascular proteins (Table 3).

Table 3

Protein function of fibrotic and angiogenic proteins.

	Protein Function	Disease implications
Fibrotic Markers
PAI-1Plasminogen Activator Inhibitor 1	Enhances ECM deposition by blocking MMP activationStimulates clot formation by inhibiting fibrinolysis [10]	Implicated in many pathologies including tissue fibrosis, obesity, cardiovascular disease [10, 11]
TIMP-1Tissue Inhibitor of Matrix Metalloproteinase 1	Restricts ECM proteolysis by inhibitingactivity of MMPs, including MMP-1, MMP-3 and MMP-9 [12]	Elevated in pulmonary, myocardial and hepatic fibrosis, and hepatitis C liver disease [13–17]
TIMP-2Tissue Inhibitor of Matrix Metalloproteinase 2	Inhibits activity of MMPs, including MMP-2 [12]	Elevated in hepatic fibrosis and hepatitis C liver disease [13, 17]
Vascular Markers
HGFHepatocyte Growth Factor	Stimulates angiogenesis by promoting proliferation, migration and survival of endothelial cellsInhibits fibrosisRegulates inflammationTissue regeneration [18, 19]	Decreased in COPDSuggested as treatment to: inhibit fibrosis, enhance tissue regeneration, treat ischemic diseases [19–21]
VEGF-AVascular Endothelial Growth Factor A	Important regulator of vascular health by stimulating development and maintenance of blood vessels [22]	Therapeutic target to stimulate or suppress angiogenesis (e.g. cancer, diabetic retinopathy) [22]

This study identified altered levels of three fibrotic markers (PAI 1, TIMP1 and TIMP2), as well as two vascular markers (HGF and VEGF-A). While we classified these proteins as pro-fibrotic or vascular, there is crosstalk between these pathways. In normal tissue, the homeostasis of extracellular matrix (ECM) is maintained through a balance between production and breakdown of collagens. Disruption of this homeostasis is observed when increased production of ECM is necessary to restore damaged tissue. Chronic tissue insult (e.g. hypertension, liver disease or diabetes), and subsequent repetitive need for tissue repair, can result in excessive accumulation of ECM, leading to tissue fibrosis. In the bladder, fibrosis thickens the bladder wall and alters its urodynamic compliance [23]. We identified increased levels of three proteins (PAI 1, TIMP1 and TIMP2) that can support a pro-fibrotic environment.

Plasminogen activator inhibitor 1 (PAI 1), or Serpin E1, inhibits the activity of urokinase-type/tissue type plasminogen activator (uPA/tPA) and plasmin. Hereby, PAI 1 indirectly inhibits the activation of plasminogen-dependent matrix metalloproteinases, such as MMP2 and MMP9, resulting in hindrance of the proteolysis of ECM and stimulation of fibrosis. As such, sustained high levels of PAI 1 are associated with fibrosis in various organs, e.g. heart, skin, liver, kidney and lung [10, 24]. Through the inhibition of uPA/tPA, PAI 1 also inhibits fibrinolysis hereby stimulating blood clot formation [25]. Thus PAI 1 could have a dual role in RC: 1) promote bladder fibrosis, 2) stimulate blood clot formation to help repair damaged blood vessels and arrest bleeding. PAI 1 levels are dependent on various factors, such as body composition and time of day; PAI 1 levels follow a circadian rhythm with highest levels in the morning [10]. In this study, we collected urine samples at random time points of the day, which can explain the large variability observed between samples.

Similar to PAI 1, tissue inhibitor of matrix metalloproteinase-1 and 2 (TIMP1 and TIMP2) can each block various MMPs from degrading the ECM [12, 26]. TIMP1 is used, in conjunction with 2 other proteins, in the Enhanced Liver Fibrosis test (ELFTM; Siemens Healthineers), which determines a patient’s risk for liver fibrosis [27]. Although elevated levels of TIMP1 in serum is an indicator of liver fibrosis, there is conflicting data from studies using pre-clinical models on how and if TIMP1 functionally contributes to tissue fibrosis [26]. Similar to liver fibrosis, our study suggests that TIMP1 could be a marker for bladder fibrosis/RC. TIMP2 has not been widely studied, though in a chemical model of liver fibrosis TIMP2 was shown to be pro-fibrotic [28]. It is feasible that TIMP1 and TIMP2 have a synergistic effect on fibrosis by inhibiting MMPs together.

As we previously reported and discussed, we identified elevated levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A) in urine of irradiated patients with RC diagnosis or symptoms [8]. VEGF-A is a master regulator of vascular homeostasis and angiogenesis. It can be produced by a large variety of cells and primarily binds its receptors on vascular and lymphatic endothelial cells to promote vasodilation, angiogenesis, and vascular permeability and homeostasis [29]. VEGF-A is increased in response to hypoxia, thus elevated levels of VEGF could indicate the presence of hypoxic tissue in the bladder. Like VEGF, HGF stimulates angiogenesis by inducing cell migration, proliferation, survival and morphogenesis [20]. However, HGF has also been shown to protect against pulmonary fibrosis. Thus, HGF could potentially play a dual role in RC whereby it promotes angiogenesis and is protective against fibrosis.

Most of the inflammatory proteins were below detectable range of the assay. The absence of pro-inflammatory proteins in the urine of RC patients is striking, given that inflammatory proteins are elevated in urine samples of patients with other bladder conditions, including interstitial cystitis, bacterial cystitis, bladder cancer, and urinary tract infections [6, 30, 31]. The cytokines IL-1a, IL-6, IL-8 and MIP-1a were detectable in one or two RC patients with gross hematuria and high symptom scores. Likewise, pre-clinical models of RC have not demonstrated a chronic inflammatory response to radiation [32-36]. One study did identify elevated levels of macrophage migration inhibitory factor (MIF) in patients with RC. Though MIF levels were compared to healthy individuals without radiation therapy history and thus we cannot conclude that these changes were radiation-induced or specific to patients with RC [37]. Thus, our findings suggest that inflammation may not play a prominent role during RC disease progression. Rather, bladder inflammation in RC patients may be indicative of end stage disease.

This study has several limitations. First, the proteins analyzed in this study were limited to the analytes available for use on the Multiplex Luminex system; other inflammatory, fibrotic or vascular proteins could be increased in the urine as well. Second, urinary protein levels might not fully reflect bladder pathology; no detectable change in the urine does not mean that analytes are not altered in the bladder itself. Third, the data is based on diagnosis and symptoms at time of urine collection; patients without symptoms at time of sample collection could develop RC-related symptoms at a later time point. This means that ongoing bladder changes could already demonstrate higher proteins levels in the urine without obvious functional bladder changes (e.g. hematuria, nocturia). Finally, we acknowledge that statistical significance at 5% level was not obtained in all comparisons. The Welch T-test was used to correct for high variance in the data, which compromises the p-value. Comparing protein changes over time within one individual through a longitudinal study would likely yield less variability in the data.

Conclusion

This is the first study that extensively explored changes in inflammatory, fibrotic and vascular proteins in urine of PCa survivors with radiation therapy history as a means to understand radiation damage to the bladder. Our data suggests that RC and associated symptoms are primarily driven by fibrosis and vascular damage and/or remodeling. Lack of changes in inflammatory cytokines suggests that inflammation is not a key characteristic of RC and thus should not be the primary target for treatment, unless for patients that are suffering from end-stage disease. Furthermore, many studies have shown that urinary-based biomarkers have high sensitivity and specificity in the diagnosis of bladder diseases (e.g. bladder cancer), thus the findings of this study provide initial evidence that urinary biomarkers might help predict, at an early stage, who will develop RC.

Patient questionnaire–Spanish.

After providing written informed consent, all participants completed this survey to collect information on patient demographics, prostate cancer history, radiation therapy, and bladder health.

(PDF)

Click here for additional data file.

Patient questionnaire–English.

English translation of the patient questionnaire.

(PDF)

Click here for additional data file.

4 Aug 2020

PONE-D-20-20879

Prostate cancer survivors with symptoms of radiation cystitis have elevated fibrotic and vascular proteins in urine

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Reviewer #1: Authors have sought to use urine analysis to investigate biomarkers for fibrosis in the bladder wall of radiation cystitis patients. Following concerns were noted

1) Experiments are predicated on the assumption that urinary elevation of (PAI 1, TIMP1 and TIMP2) proteins can serve as liquid biopsy for fibrosis. The underlying assumption is missing support from either urodynamic data or bladder wall histology to support a causal relationship between increased levels of three proteins (PAI 1, TIMP1 and TIMP2) and a pro-fibrotic environment in RC patients with hematuria.

2) Since hematuria is not unique to RC, authors should qualify the specificity of their findings with respect to other disorders exhibiting hematuria.

3) When authors claim that urinary proteins are stable for up to one year at room temperature with norgen preservative, what is the rationale for exposing urine samples to freeze thaw cycle before analysis. Did the authors consider analyzing urine samples directly without freeze thaw to rule out whether extensive processing of urine samples containing norgen preservative followed by centrifugation, freezing at -80 and thawing contributes to the striking absence of pro-inflammatory proteins in the urine of RC patients. Or is the lack of detection of urinary inflammatory proteins due to protein degradation during interval between collection and analysis.

4) The claim "inflammation is not the driving factor of the disease" in patients is speculative and not backed by any supportive evidence.

5) Is it correct that hematuria was seen in only 5 of the RC patients as per table 1. It was hard to link the number in table 1 with fig. 2 scatter plot for hematuria.

6) Are the p values corrected for multiple testing

7) More details of RC diagnosis needs to be added as "decreased maximal bladder capacity as measured during cystoscopy" does not make sense

Reviewer #2: This is in interesting well written paper looking at urinary levels of various urinary proteins to determine possible areas of abnormality to guide future therapy.

Page 9 line 59 - surely this should read " urinary diversion with or without cystectomy" not vice versa?

Page 10 line 73 - should read 'but are' not 'but is'

Page 11 line 85 - why are the patients from Chile only?

Page 11 line 90 - what was the maximal bladder capacity in no radiation, radiation with no radiation cystitis and radiation cystitis. The definition of radiation cystitis is vague and needs clarity.

Page 11 line 98 - what symptoms cires were used? It looks very much that this is a 'home made' symptom score. What was the reason for using this as opposed to the many validated and international recognised symptom scores such as IPSS and ICIQ OAB

Page 11 line 99 - likewise on what basis was is 10 selected as a cut off for the severe symptoms?

Page 11 line 105 - whta is th eevidence that storage/freezing and shipping didn't affect urinary protein levels?

Page 12 line 115 - how do the protein levels in those with IC compare to those with RC?

Page 13 Table - please add an addendum defining the markers and explaining their putative actions

Page 15 Table 2 - do you have urodynamic parameters or a frequency/volume chart to help with your definitions they are all somewhat vague?

Page 15 Table 2 - I don't think GU spasm is a recognised term - please can you define.

Page 21 line 281 - perhaps the samples would have been better collecting all at the same time ie 1st void of the day or 0900? Please comment on this and other factors that may affect urinary proteins - diet/hydration/medications - how can (and should they be) controlled for ?

Page 22 conclusions - Is significance the correct test for relevance of urinary proteins/biomarkers? Could there be a threshold phenomena - please discuss.

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2 Oct 2020

Thank you for the constructive feedback. We have made changes to the improved manuscript and hope that this is now acceptable for publication.

Reviewer #1: Authors have sought to use urine analysis to investigate biomarkers for fibrosis in the bladder wall of radiation cystitis patients. Following concerns were noted

1) Experiments are predicated on the assumption that urinary elevation of (PAI 1, TIMP1 and TIMP2) proteins can serve as liquid biopsy for fibrosis. The underlying assumption is missing support from either urodynamic data or bladder wall histology to support a causal relationship between increased levels of three proteins (PAI 1, TIMP1 and TIMP2) and a pro-fibrotic environment in RC patients with hematuria.

While we agree with the reviewers point, we did not perform bladder wall histology on these patients. Taking biopsies of bladders with RC can result in severe complications for the patients and most patients will not consent for this. We did not collect urodynamic data for all participants. We attempt to determine maximal bladder capacity during cystoscopy in patients that are suspected to have RC, though due to the risk of tearing of fragile blood vessels, maximal bladder capacity is often not reached. We did compare frequency data with urinary levels of pro-fibrotic factors, but this yielded no significant results.

2) Since hematuria is not unique to RC, authors should qualify the specificity of their findings with respect to other disorders exhibiting hematuria.

All patients with micro- or macrohematuria were studied with cystoscopy for confirmation of radiation cystitis and to rule out other causes of hematuria. We added this information to the manuscript to clarify. Thank you.

3) When authors claim that urinary proteins are stable for up to one year at room temperature with norgen preservative, what is the rationale for exposing urine samples to freeze thaw cycle before analysis. Did the authors consider analyzing urine samples directly without freeze thaw to rule out whether extensive processing of urine samples containing norgen preservative followed by centrifugation, freezing at -80 and thawing contributes to the striking absence of pro-inflammatory proteins in the urine of RC patients. Or is the lack of detection of urinary inflammatory proteins due to protein degradation during interval between collection and analysis.

For the urine samples both preservative and freezing were used for several reasons:

1. The preservative was used to allow for short term storage in the clinic and shipping of the samples at room temperature. This allowed for collection of a greater number of samples at the clinic before shipping, hereby minimizing the number of necessary shipments. This prevented possible freeze/thaw accidents due to hold up of samples at customs. Also, at the clinic they do not have direct access to a centrifuge to remove any debris in the urine, nor was there access to a -80C. If preservative is not used, urine samples need to be frozen down immediately after collection to avoid degradation of proteins/RNA.

2. Urine samples were collected over 3 years, and the preservative is only stable for up to 1 year.

3. To avoid inter-experimental variation, we ran all the urine samples at the same time. Thus, we needed to wait until all the samples were collected.

4. Multiplex Luminex plates are costly; thus we choose to only run full plates.

The process of using preservative and freezing the urine samples was also used by our group for our interstitial cystitis biomarker studies. In these studies we did find detectable and elevated levels of cytokines in the urine. In addition, several urine samples from patients with severe RC did contain high levels of cytokines, as we reported in this manuscript. Thank you.

4) The claim "inflammation is not the driving factor of the disease" in patients is speculative and not backed by any supportive evidence.

We agree with the reviewers and have altered the language in the manuscript. Thank you.

5) Is it correct that hematuria was seen in only 5 of the RC patients as per table 1. It was hard to link the number in table 1 with fig. 2 scatter plot for hematuria.

15 of the urine samples we collected were from patients that had been diagnosed with RC. Of these 15, 9 reported hematuria. Of the 9 RC patients with hematuria, 5 reported gross hematuria with blood clots.

6) Are the p values corrected for multiple testing

No, the p-values are not corrected for multiple testing as only the means of 2 groups were tested in each one-way ANOVA test. Thank you.

7) More details of RC diagnosis needs to be added as "decreased maximal bladder capacity as measured during cystoscopy" does not make sense

The definition we used is the classical definition of RC: pale bladder mucosa and telangiectasia. The bladder has fragile vessels that bleed easily during the endoscopy. If possible, we tried to fill the bladder to its maximum capacity, but because of the danger of inducing more bleeding, this is often not feasible. Most patients with macrohematuria receive a bladder catheter in the emergency room before they are admitted in the department of Urology to remove blood clots and to irrigate the bladder. When a cystoscopy is performed afterwards, we find erosion of the bladder mucosa and edema related to the use of the catheter.

Reviewer #2: This is in interesting well written paper looking at urinary levels of various urinary proteins to determine possible areas of abnormality to guide future therapy.

Page 9 line 59 - surely this should read " urinary diversion with or without cystectomy" not vice versa?

We apologize for the confusion. With urinary diversion we were eluting to the reconstruction of a neobladder. We have corrected this in the manuscript.

Page 10 line 73 - should read 'but are' not 'but is'. – this grammatical error was corrected.

Page 11 line 85 - why are the patients from Chile only?

This study was done in collaboration with Dr. Heinz Nicolai, a urologist from the University of Chile in Santiago de Chile. He was responsible for patient recruitment and urine collection. Blinded urine samples were subsequently shipped to Beaumont, processed and analyzed. RC is considered an orphan disease and recruiting a large number of patients without introducing too many variables (e.g. ethnicity, radiation dose, treatment strategy, type of pelvic cancer, gender, age) is challenging. To minimize the number of variables, we recruited only men with a history of prostate cancer that received external beam radiation therapy at one location. Future studies will focus on analyzing urine samples from patients from different locations, and with different cancer history (e.g. cervical cancer, colorectal cancer).

Page 11 line 90 - what was the maximal bladder capacity in no radiation, radiation with no radiation cystitis and radiation cystitis. The definition of radiation cystitis is vague and needs clarity.

No urodynamic measurements were performed in all participants. As mentioned earlier, we attempt to determine the maximal bladder capacity in patients that are suspected to have RC and that receive a cystoscopy. However, due to the fragile nature of the bladder vasculature, reaching maximal bladder capacity is often not feasible. Therefore, we cannot provide you with maximal bladder capacity for the different groups.

As for the definition of radiation cystitis: this is the classical way in which RC is diagnosed using cystoscopy. Please see also our response to reviewer #1 (Comment #7).

Page 11 line 98 - what symptoms cires were used? It looks very much that this is a 'home made' symptom score. What was the reason for using this as opposed to the many validated and international recognized symptom scores such as IPSS and ICIQ OAB.

We developed this survey specifically for RC and have used it in a previous publication [Zwaans et al. 2016 (ref 8)]. We included the reference in the revised manuscript.

Page 11 line 99 - likewise on what basis was is 10 selected as a cut off for the severe symptoms?

A score of 10 corresponds to the presence of two symptoms (hematuria, gross hematuria, incontinence, nocturia or GU spasm) occurring multiple times per day, or to 3 or more symptoms occurring at minimum once per week. We believe this is a clear indication of an underlying urological defect.

Page 11 line 105 - what is the evidence that storage/freezing and shipping didn't affect urinary protein levels?

Protein and nucleotides in urine samples are extremely vulnerable to degradation at room temperature and thus it is really important that urine samples are processed in a timely manner after collection. The standard operating procedure for urine collection and storage is: Spin down at low speed, aliquoting supernatant and storing at -80C. Here are several studies on urinary cytokines in which this process has been followed (PMID: 29891542, 20942931, 26119560). In the latter study (PMID 26119560) the researchers added a ‘homemade’ preservative to urine samples prior to storage at -80C to ensure protein stability was maximized. Urine samples were from patients with non-muscle invasive bladder cancer. They subsequently performed Multiplex Luminex assay, as we did in our study. Cytokines analyzed in this study included IL-2, IL-8, IL-6, IL-1ra, IL-10, IL-12[p70], IL-12[p40], TRAIL, and TNF-α. As an example, reported ranges for IL-6 were (0 – 2780 pg/ml) and for IL-8 (-12 – 2998 pg/ml).

For our study, we ensured that urine samples were only frozen once prior to protein analysis to minimize protein degradation due to freeze/thaw cycles.

Page 12 line 115 - how do the protein levels in those with IC compare to those with RC?

We have not made a direct comparison between IC samples and RC samples of all the analytes reported in this study. Our group has reported elevated levels of IL-6, IL-8 and GRO (CXCL1) in patients with IC (with and without ulcers) in comparison to control urine (PMID: 29088231). In IC patients, IL-6 ranged from 1.4-4.4 pg/ml, IL-8 ranged from 20-54 pg/ml, and GRO ranged from 10-21.5 pg/ml. For RC samples in which IL-6 and IL-8 were detected, IL-6 levels were on average 2.1 pg/ml and IL-8 levels were on average 20 pg/ml. But it is important to note that these two cytokines were only detected in a small portion of samples.

Page 13 Table - please add an addendum defining the markers and explaining their putative actions. A third table has been added with this information

Page 15 Table 2 - do you have urodynamic parameters or a frequency/volume chart to help with your definitions they are all somewhat vague?

No, we do not have urodynamic parameters available. We did collect frequency data that is documented in Table 2. In the early stages of RC, we often find an increase of their void frequency, later in an advanced state of RC, we clearly observe pollakiuria, nocturia and urgency.

Page 15 Table 2 - I don't think GU spasm is a recognized term - please can you define.

We changed this to Bladder spasm. We agree that the terminology of spasm can be inappropriate here since we generally observe the spasm in patients with RC that require a bladder catheter.

Page 21 line 281 - perhaps the samples would have been better collecting all at the same time i.e. 1st void of the day or 0900? Please comment on this and other factors that may affect urinary proteins - diet/hydration/medications - how can (and should they be) controlled for?

Collecting urine at the first void of the day could help in tightening the data for certain factors such as PAI-1. However, our goal is to identify urine biomarkers that are robust enough so they are not sensitive to the time at which urine was collected. In addition, we have

Page 22 conclusions - Is significance the correct test for relevance of urinary proteins/biomarkers? Could there be a threshold phenomena - please discuss.

We agree that a threshold for urinary protein levels rather than a significance level could be more relevant with respect to identifying potential urinary biomarkers for RC. However, in this study, we believe that using a significance test is more appropriate. First, the goal of the study was to use urine samples as “liquid biopsies” to identify proteins that play a role in the disease progression and not per se to identify protein biomarkers. A follow-up study could look into which of these proteins could function as possible biomarkers. Second, to set a clear threshold we will have to increase the number of samples, especially for proteins with small concentration ranges, and collecting samples at same time of day to minimize data variability would be needed.

There is a fair amount of inter-subject variability with respect to levels of the tested proteins in the urine. We believe that the best method to screen for the development of RC using urine, is to track the concentration of several factors over time within one individual. But this idea would need to be tested using a longitudinal study.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

14 Oct 2020

Prostate cancer survivors with symptoms of radiation cystitis have elevated fibrotic and vascular proteins in urine

PONE-D-20-20879R1

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Authors have responded constructively to earlier comments. They should consider following recommendations

Since all the significant results in Fig.2 and 3 are linked with the symptom of hematuria and the urodynamic data is missing, authors should emphasize on hematuria symptom in the title and text of the manuscript .

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Reviewer #1: No

16 Oct 2020

PONE-D-20-20879R1

Prostate cancer survivors with symptoms of radiation cystitis have elevated fibrotic and vascular proteins in urine

Dear Dr. Lamb:

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