Purpose: INGN 201 (Ad-p53) is a replication-defective adenoviral vector that encodes a wild-type p53 gene driven by the cytomegalovirus promoter. INGN 201 has been shown to have antitumoral activity against human prostate cancer cell lines. This study was undertaken to determine the safety of INGN 201 in patients with locally advanced prostate cancer, to assess transgene expression, and to evaluate antitumoral activity.

Experimental Design: Our study included patients with clinical stage T3, T1c-T2a with Gleason score 8–10 disease, or T2a-T2b with Gleason score 7 disease and a prostate-specific antigen level >10 ng/ml. INGN 201 was administered by intraprostatic injection under ultrasonographic guidance. One course of INGN 201 was defined as three separate INGN 201 administrations 2 weeks apart. Biopsies at baseline and 24 h after the first administration were assessed for p53 protein by immunohistochemical staining and for apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay.

Results: A total of 38 courses of INGN 201 gene therapy were administered to 30 patients, of whom 26 underwent radical prostatectomy. There were no grade 3 or 4 adverse events related to INGN 201 administration. Of the 11 patients with negative baseline immunostaining for p53 protein, 10 had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in apoptotic cells by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. All 26 of the patients who underwent radical prostatectomy had significant residual viable prostate cancer, and 12 have experienced biochemical failure (median follow-up, 42 months).

Conclusion: Intraprostatic INGN 201 gene therapy is safe and can reliably result in p53 protein production and apoptosis.

Approximately 10% of patients diagnosed with prostate cancer have locally advanced disease and are at high risk of disease progression despite local therapy with radical prostatectomy or radiation therapy. These patients can be accurately identified based on their clinical stage, tumor grade, and initial prostate-specific antigen (PSA) level, but the optimal treatment for this group remains controversial. Because external beam radiation therapy and radical prostatectomy used alone have significant limitations in their ability to eradicate locally advanced prostate cancer, interest has shifted toward the use of multimodality therapy. In the development of multimodality approaches involving radical prostatectomy, to date, there has been no evidence of any improvement in biochemical outcome for patients treated with hormonal therapy plus surgery when compared with surgery alone. Because standard therapies have poor results, we believe that patients with locally advanced prostate cancer are excellent candidates for experimental therapies including gene therapy.

The p53 gene has been extensively studied and is known to play a critical role in cell cycle regulation and control of apoptosis (1). The p53 protein is a multifunctional protein that can act as a transcriptional activator or repressor, is induced by DNA damage, and interacts with proteins involved in DNA replication and repair (2). The p53 gene appears to have an important role in sensing and repairing DNA damage, inhibiting the cell cycle to allow DNA repair, and inducing apoptosis to eliminate severely damaged cells (2). Alterations in the p53 gene play an important role in the progression of human prostate cancer (3). Overexpression of the p53 protein has been shown to be an independent predictor of disease-free and overall survival after surgery (4, 5) or radiation therapy (6, 7) in patients with prostate cancer. Most studies have reported a low frequency (4–20%) of p53 alterations in primary prostate tumors (8, 9). In contrast, when samples of metastatic tumors are included, particularly samples of bone metastases, the frequency of p53 alterations increases to 50–79% (10, 11, 12). Both Navone et al. (13) and Stapleton et al. (3) have demonstrated clonal expansion of p53 mutations from the primary tumor to metastases in paired samples of primary cancers and metastases from the same patients. Taken together, these results suggest that foci of p53 mutants in the primary tumor may have a selective advantage and a higher metastatic potential.

An adenoviral vector system was selected for gene therapy because of its ability to infect many cell types, including quiescent and dividing cells, without integration into the host genome, because of its high-level transient expression and capacity to be produced at high titers, and because of the reported safety of adenoviral vaccines (14). INGN 201 (Ad-p53) is a replication-impaired adenoviral vector that encodes a wild-type p53 gene driven by the cytomegalovirus promoter. Preclinical studies with INGN 201 have shown that p53 transduction can induce apoptosis and decrease cell proliferation in a number of cancer cell lines without adversely affecting normal cells (15, 16, 17). Importantly, p53 gene therapy is active against cancer cells expressing wild-type or mutated p53(18, 19). INGN 201 reduces tumor growth in xenograft models of prostate cancer and other malignancies (20, 21, 22, 23, 24). In model systems, INGN 201 has also been shown to enhance the antitumoral activity of chemotherapy and radiation therapy (24, 25, 26).

This study was undertaken to determine the safety of INGN 201 in patients with locally advanced prostate cancer, to evaluate transgene expression, and to assess antitumoral activity. The feasibility of direct intraprostatic injections of INGN 201 was investigated in patients with locally advanced prostate cancer in a neoadjuvant setting before radical prostatectomy.

Protocol Approval.

The protocol used in our study was approved by the Biosafety Committees and the Surveillance Committees/Institutional Review Boards of the participating institutions, the Recombinant DNA Advisory Committee of the NIH, and the United States Food and Drug Administration. Written informed consent was obtained from all of the patients stating that they were aware of the investigational nature of this study, in keeping with institutional policies.

Gene Transfer Vector.

INGN 201 (Advexin) was supplied by Introgen Therapeutics, Inc. (Houston, TX) in frozen aliquots containing 1 × 1012 viral particles per ml in PBS containing 10% glycerol. Construction and generation of the vector was reported previously (27).

Eligibility Criteria and Treatment Protocol.

The study was limited to patients with histologically confirmed prostate adenocarcinoma and no clinical evidence of metastasis (negative bone scan and computed tomography of the pelvis). Patients were eligible for inclusion in the study if their cancers were clinically staged as T3, T1c-T2a with Gleason score 8–10 disease, or T2a-b with Gleason score 7 disease and a PSA level >10 ng/ml. Patients with these inclusion criteria are unlikely to be cured with surgery alone. Patients were required to have a surgically resectable prostate and a 10-year life expectancy. No prior therapy for prostate cancer was allowed. Because INGN 201 has been shown to have antitumoral activity in cancer cells expressing wild-type p53, study participants were not required to have evidence of p53 protein overexpression in pretreatment tumor biopsy samples.

All of the patients had measurable lesions on baseline transrectal ultrasonography (TRUS) or magnetic resonance imaging (MRI) before treatment, and underwent repeat TRUS and MRI after each course of INGN 201 gene therapy. Lesions seen on TRUS or MRI were only considered positive if they corresponded to a region of the prostate shown to have cancer on biopsy. One course of INGN 201 therapy was defined as three separate INGN 201 administrations 2 weeks apart. Patients who demonstrated a clinical response to INGN 201 (≥25% reduction in volume of visible lesions on TRUS or MRI) after one course were treated with additional courses of INGN 201. We estimated tumor volumes by calculating the product of the perpendicular diameters of the indicator lesion seen on TRUS or MRI. Patients could receive up to three courses of INGN 201 before radical prostatectomy. Each administration of INGN 201 consisted of injecting 3 ml of INGN 201 divided equally among five to six transperineal percutaneous injection sites. The INGN 201 administrations were performed under transrectal ultrasonographic guidance with the patient under monitored sedation. At each injection site, the needle was inserted to the base of the prostate, and INGN 201 was injected as the needle was withdrawn toward the apex of the prostate. The goal was to encompass the entire prostate with INGN 201. All of the patients underwent prostate biopsies 1 day after the first administration of INGN 201 to assess gene transfer. The doses of INGN 201 were escalated between patient groups from 3 × 1010 viral particles to 3 × 1012 viral particles, with escalations in one half or one log increments. Between 3 and 18 patients were assigned to each dose level. All 30 of the patients in the study received INGN 201, and 26 of 30 patients underwent radical prostatectomy 2 weeks after the final course of INGN 201. The toxic effects of therapy were evaluated according to National Cancer Institute toxicity criteria (28).

Patients were followed postoperatively at 3-month intervals with serial PSA determinations. No additional therapy was given until evidence of biochemical failure, defined as any detectable PSA level after radical prostatectomy or as two serial rises in PSA level in a patient who did not undergo radical prostatectomy.

Tissue Samples for Analysis of Gene Expression and Apoptosis.

Patients were selected for inclusion in immunohistochemical analyses and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays by review of biopsy samples obtained before treatment and 24 h after the first administration of INGN 201. If sufficient tumor cells were appreciated in the same geographical area in both sets of samples from a patient, the samples were earmarked for analysis.

Immunohistochemistry.

To determine p53 protein status, tissue sections were immunostained using p53 antibody D07 (Dako) as the primary antibody. Tissue preparation has been described previously (12). A biotinylated Universal Secondary Antibody (Dako) was applied followed by horseradish peroxidase-labeled streptavidin (Dako; K0690). Events were visualized using 3,3′-diaminobenzidine substrate (Dako; K3466). A specimen was scored as positive if at least 5% of the tumor cells showed nuclear staining.

TUNEL Assay for DNA Fragmentation.

To assess apoptosis, tissue sections for TUNEL analysis were processed as described previously (29). The apoptotic index was determined by scoring the TUNEL-positive tumor cells and expressing the value as a percentage of 500 tumor cells in the tissue section. The immunohistochemistry and TUNEL sections were each coded and read by a single observer who had no knowledge of the patients, biopsy sequence, or clinical status.

Patient and Tumor Characteristics.

Thirty patients were enrolled in the study, and their clinical characteristics are summarized in Table 1. A total of 38 courses (114 separate administrations) of INGN 201 gene therapy were administered to these 30 patients (1 course in 23 patients, 2 courses in 6 patients, and 3 courses in 1 patient). The median follow-up was 19 months. Twenty-six patients underwent a radical prostatectomy after the final course of INGN 201; in 3 patients, the planned radical prostatectomy was aborted because of intraoperative findings of nodal metastasis, and 1 patient withdrew consent before surgery.

Assessment of Gene Transfer and Apoptosis.

Prostate samples of 19 patients were suitable for immunohistochemical analysis and TUNEL assay (Table 2). Positive immunostaining for p53 in the untreated samples presumably reflects cells with mutant p53, whereas negative samples are assumed to express wild-type p53. Of the 11 patients with negative baseline immunostaining for the p53 protein, 10 had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in TUNEL staining. In total, 16 of the 19 patients had positive immunostaining for the p53 protein 24 h after the first administration of INGN 201, and 12 patients had increased TUNEL staining. Representative samples are shown in Fig. 1. The mean pretreatment apoptotic index was 0.39 with a SD of 0.25. The mean post-treatment apoptotic index was 1.18 with a SD of 1.25. A one-sided Wilcoxon signed-rank test showed that the post-treatment apoptotic index was significantly higher than the baseline apoptotic index (P = 0.002; Fig. 2).

Clinical Results.

Serum PSA measurements before and after INGN 201 administration were available in 28 patients. There was no significant change between the baseline and post-INGN 201 gene therapy serum PSA (P = 0.65, Wilcoxon rank-sum test). The pathological findings in the 29 patients who underwent pelvic lymph node dissection and 26 patients who underwent radical prostatectomy are summarized in Table 3. All 26 of the patients who underwent radical prostatectomy had significant residual viable prostate cancer. We observed a moderate to intense inflammatory cell infiltrate in 14 of the radical prostatectomy specimens, consistent with other reports of immune-mediated responses after adenovirus treatment (30). There was no clear relationship between the degree or extent of inflammation and the viral dose or number of courses of gene therapy. Of the 26 patients who underwent radical prostatectomy, 12 have experienced biochemical failure with a median follow-up of 42 months. The biochemical disease-free survival for the 26 patients undergoing radical prostatectomy is shown in Fig. 3. Of the 4 patients who did not undergo radical prostatectomy, 1 has had a rising PSA level, and the other 3 were lost to follow-up.

Adverse Events.

All 30 of the patients who underwent INGN 201 gene therapy were evaluable for adverse events (Table 4). There were no treatment-related or disease-related deaths during the study period. Fever, headache, chills, and perineal pain at the injection site were the most common adverse events of INGN 201 administration. The fever was generally self-limiting and easily treated with acetaminophen. No grade 3 or 4 adverse events were noted.

The 26 patients who underwent radical prostatectomy did not experience any unusual surgical complications. No patient developed a wound infection, seroma, or lymphocele. One patient (4%) had a rectal injury that was closed primarily without sequelae, 1 patient (4%) had a transient urine leak postoperatively, and 5 patients (19%) had long-term stress urinary incontinence requiring pad use.

There is mounting evidence of the importance of the p53 gene in the progression of human prostate cancer. On the basis of preclinical evidence of the antitumor activity of INGN 201 in human prostate cancer models containing wild-type or mutated p53, we opted to evaluate this treatment in a neoadjuvant setting before radical prostatectomy in patients with locally advanced prostate cancer.

One important finding of our study was that multiple doses of INGN 201 could be safely administered by intraprostatic injection. Our study was a dose-escalation study, and because of the lack of serious adverse events, we were able to rapidly increase to the highest dose level allowed by our protocol, 3 × 1012 viral particles. No grade 3 or 4 adverse events related to INGN 201 administration were seen. The most common grade 1 and 2 adverse events were fever, headache, chills, and perineal pain at the injection site; the fever was treated with acetaminophen and resolved within 24–48 h in most cases. Similarly, pain at the injection site and fever were the most common adverse events reported by Clayman et al. (31) and Swisher et al. (32) with INGN 201 injection into head and neck cancers and lung cancers, respectively. Due to the lack of serious adverse events, most of our patients were treated on an outpatient basis. The minimal adverse effects of INGN 201 may allow it to be used in combination with conventional treatments such as radiation therapy or hormonal therapy.

In our study, we evaluated p53 gene expression and the desired biological effect of apoptosis by comparing p53 immunostaining and TUNEL results in prostate needle biopsies before and 24 h after INGN 201 administration. Prostate biopsy tissue is sparse, and we attempted to reduce the potential influence of sampling error by limiting our analysis to samples of histologically similar tumor obtained from the same location of the prostate. Despite these efforts, sampling error remains a potential limitation of our report. The clearest evidence for successful INGN 201 gene expression was seen among the 11 patients with negative baseline immunostaining for the p53 protein. Ten of these patients had positive p53 immunostaining after the first administration of INGN 201, and 8 had an increase in TUNEL staining of cancer cells. We opted to perform biopsies 24 h after the first INGN 201 administration to make the procedure convenient for patients and to retrieve tissue samples at a time when gene expression should be high. Data in preclinical models, including in vivo models, indicate that apoptosis is induced 24 h after administration of INGN 201 at a level far beyond that seen with a control adenoviral vector (23, 24). Detection of gene expression in vivo after completion of INGN 201 gene therapy may be difficult because successful transfer and expression of wild-type p53 in a tumor may result in rapid apoptosis and cell death. One limitation of our study is that we did not use an empty control vector; therefore, we cannot exclude that the viral infection per se or the injection procedure itself, was responsible for this increased apoptosis. We believe that our TUNEL assay findings indicate that proapoptotic pathways are present in locally advanced prostate cancer and are a therapeutically exploitable target.

Our study is, to our knowledge, the first to involve targeted gene therapy to an organ followed by surgical extirpation. Radical prostatectomy in our patients was performed 2 weeks after the last administration of INGN 201, at a time when gene expression is known to be reduced and pathological evidence of antitumoral activity should be optimally detectable. All of the patients had significant residual areas of viable carcinoma, and no areas of widespread cell destruction were noted. Thus, INGN 201 gene therapy alone is insufficient to eradicate locally advanced prostate cancer (at least by this schedule of administration), and this therapy may have greater antitumoral activity when used in combination with other proapoptotic treatments, such as systemic chemotherapy, hormonal therapy, or radiation therapy. Preclinical studies suggest synergistic activity between INGN 201 and systemic chemotherapy (25), and INGN 201 and radiation therapy (24, 26)in vitro. In the study by Cowen et al. (24), prostate tumor growth in vivo was inhibited supra-additively when p53null and p53wild-type tumors were treated with INGN 201 and 5 Gy radiation.

The biochemical disease-free survival of patients treated with INGN 201 and radical prostatectomy is similar to the biochemical disease-free survival of similar staged locally advanced patients treated with systemic chemotherapy followed by radical prostatectomy (33). It is conceivable that the biochemical results of patients treated with INGN 201 in a preoperative setting might improve if INGN 201 was used in combination with other proapoptotic stimuli such as hormonal therapy or systemic chemotherapy.

The main treatments for prostate cancer, surgery, radiation therapy, and hormonal therapy, have been around for well over 50 years, and there is a desperate need for new therapeutic strategies. Prostate-targeted gene therapy offers hope, with a variety of biological mechanisms that might be exploited. In our study, we demonstrated that INGN 201 can be safely administered by intraprostatic injection and that intraprostatic injection of INGN 201 reliably results in p53 protein production and apoptosis. Our findings indicate that the molecular pathways for apoptosis are present in locally advanced prostate cancer and can be therapeutically exploited using gene therapy. Furthermore, INGN 201 activity may be enhanced, to fully exploit proapoptotic pathways, when combined with radiation therapy, hormonal therapy, or systemic chemotherapy.

Grant support: American Cancer Society Grants RPG96-036-04-CDD and RSGCDD-10154, the Assisi Foundation, Hyde Family Foundation, NIH Prostate Specialized Programs of Research Excellence P50 CA90270, and CapCure Grant 80095069.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Louis L. Pisters, Department of Urology, Unit 446, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3250; Fax: (713) 794-4824; E-mail: lpisters@mdanderson.org

Fig. 1.

Immunohistochemical staining for p53 protein and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining for apoptotic cells before and 24 h after first administration of INGN 201. A, negative baseline p53 immunostaining. B, positive immunostaining for p53 protein after INGN 201 administration. C, baseline TUNEL assay. D, TUNEL assay showing increased apoptosis after INGN 201 administration.

Fig. 1.

Immunohistochemical staining for p53 protein and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining for apoptotic cells before and 24 h after first administration of INGN 201. A, negative baseline p53 immunostaining. B, positive immunostaining for p53 protein after INGN 201 administration. C, baseline TUNEL assay. D, TUNEL assay showing increased apoptosis after INGN 201 administration.

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Fig. 2.

Actual baseline and post-treatment apoptotic indices in 19 patients undergoing INGN 201 gene therapy. P = 0.002, Wilcoxon signed rank test.

Fig. 2.

Actual baseline and post-treatment apoptotic indices in 19 patients undergoing INGN 201 gene therapy. P = 0.002, Wilcoxon signed rank test.

Close modal
Fig. 3.

Biochemical disease-free survival for 26 patients undergoing radical prostatectomy after INGN 201 gene therapy. Biochemical failure was defined as any detectable prostate-specific antigen level after radical prostatectomy. Downward ticks represent censored patients.

Fig. 3.

Biochemical disease-free survival for 26 patients undergoing radical prostatectomy after INGN 201 gene therapy. Biochemical failure was defined as any detectable prostate-specific antigen level after radical prostatectomy. Downward ticks represent censored patients.

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Table 1

Clinical characteristics of 30 patients with prostate cancer

CharacteristicNo. of patients
Age  
 Median (yrs) 62.9 
 Range (yrs) 52–74 
Clinical T stage  
 T1c 
 T2a 17 
 T2b 
 T3a 
 T3b 
Gleason score  
 6 
 7 16 
 8 
 9 
 10 
Pretherapy serum prostate-specific antigen level  
 0–4.0 ng/ml 
 4.1–10.0 ng/ml 10 
 10.1–20.0 ng/ml 
 >20.0 ng/ml 
CharacteristicNo. of patients
Age  
 Median (yrs) 62.9 
 Range (yrs) 52–74 
Clinical T stage  
 T1c 
 T2a 17 
 T2b 
 T3a 
 T3b 
Gleason score  
 6 
 7 16 
 8 
 9 
 10 
Pretherapy serum prostate-specific antigen level  
 0–4.0 ng/ml 
 4.1–10.0 ng/ml 10 
 10.1–20.0 ng/ml 
 >20.0 ng/ml 
Table 2

Assessment of gene transfer and apoptosis in biopsy samples before and after first treatment with INGN 201

PatientPretherapyp53 ImmunostainingApoptotic Index
Clinical stageGleason scoreSerum PSAa ng/mlViral dose VPBaselinePost-treatmentBaselinePost-treatment
T2a 11.9 1 × 1011 − 0.2 1.4b 
T3a 4.4 3 × 1011 − 0.4 0.4 
T2a 17.7 3 × 1011 − 0.2 0.6b 
11 T3b 63.8 1 × 1012 − 0.2 0.4 
14 T3b 4.2 3 × 1012 − 0.4 0.8b 
16 T2a 22.5 3 × 1012 − − 0.0 0.0 
17 T2a 45.7 3 × 1012 − 0.4 3.6b 
18 T2b 21.8 3 × 1012 − 0.6 0.6 
19 T2a 10 6.2 3 × 1012 − 0.4 1.6b 
20 T3a 4.2 3 × 1012 − 0.2 2.8b 
21 T2a 13.0 3 × 1012 0.8 0.4 
22 T3a 4.4 3 × 1011 − 0.4 0.4 
23 T3b 15.5 3 × 1012 0.4 0.8b 
24 T3a 17.9 3 × 1012 − 1.0 0.6 
25 T3b 3.2 3 × 1012 0.4 0.8b 
26 T2a 5.7 3 × 1012 0.4 1.2b 
27 T3b 7.7 3 × 1012 − 0.2 0.2 
28 T2a 22.4 3 × 1012 0.4 0.4 
29         
30 T2a 5.7 3 × 1012 0.6 1.2b 
PatientPretherapyp53 ImmunostainingApoptotic Index
Clinical stageGleason scoreSerum PSAa ng/mlViral dose VPBaselinePost-treatmentBaselinePost-treatment
T2a 11.9 1 × 1011 − 0.2 1.4b 
T3a 4.4 3 × 1011 − 0.4 0.4 
T2a 17.7 3 × 1011 − 0.2 0.6b 
11 T3b 63.8 1 × 1012 − 0.2 0.4 
14 T3b 4.2 3 × 1012 − 0.4 0.8b 
16 T2a 22.5 3 × 1012 − − 0.0 0.0 
17 T2a 45.7 3 × 1012 − 0.4 3.6b 
18 T2b 21.8 3 × 1012 − 0.6 0.6 
19 T2a 10 6.2 3 × 1012 − 0.4 1.6b 
20 T3a 4.2 3 × 1012 − 0.2 2.8b 
21 T2a 13.0 3 × 1012 0.8 0.4 
22 T3a 4.4 3 × 1011 − 0.4 0.4 
23 T3b 15.5 3 × 1012 0.4 0.8b 
24 T3a 17.9 3 × 1012 − 1.0 0.6 
25 T3b 3.2 3 × 1012 0.4 0.8b 
26 T2a 5.7 3 × 1012 0.4 1.2b 
27 T3b 7.7 3 × 1012 − 0.2 0.2 
28 T2a 22.4 3 × 1012 0.4 0.4 
29         
30 T2a 5.7 3 × 1012 0.6 1.2b 
a

PSA, prostate-specific antigen; VP, viral particles;

b

Posttreatment apoptotic index above 95% confidence interval of pretreatment apoptotic index.

Table 3

Pathologic findings for 29 patients who underwent pelvic lymph node dissection and 26 patients who underwent radical prostatectomy

Pathologic findingNo. of patients%
Organ-confined 9/26 35 
Extraprostatic extension 16/26 62 
Seminal vesicle invasion 11/26 42 
Bladder neck and/or rectal invasion 0/26 
Positive lymph nodes 13/29 45 
Positive surgical margin 5/26 19 
Pathologic findingNo. of patients%
Organ-confined 9/26 35 
Extraprostatic extension 16/26 62 
Seminal vesicle invasion 11/26 42 
Bladder neck and/or rectal invasion 0/26 
Positive lymph nodes 13/29 45 
Positive surgical margin 5/26 19 
Table 4

Total number of adverse events in 30 patients after 38 courses of intraprostatic INGN 201 administration

EventGrade 1Grade 2Grade 3Grade 4Total
Perineal pain 10 15 
Fever 40 44 
Chills 14 16 
Headache 15 18 
Hematospermia 
Hematuria 
Scrotal edema 
EventGrade 1Grade 2Grade 3Grade 4Total
Perineal pain 10 15 
Fever 40 44 
Chills 14 16 
Headache 15 18 
Hematospermia 
Hematuria 
Scrotal edema 
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