Archivio Italiano di Urologia e Andrologia 2016; 88, 4304 ORIGINAL PAPER Prostatic calculi detected in peripheral zone of the gland during a transrectal ultrasound biopsy can be significant predictors of prostate cancer Lucio Dell’Atti 1, Andrea B. Galosi 2, Carmelo Ippolito 1 1 Division of Urology, University Hospital “St. Anna”, Ferrara, Italy; 2 Division of Urology, Marche Polytechnic University, Ancona, Italy. Purpose: Prostatic calculi (PC) are usually associated with benign prostatic hyperpla- sia or chronic inflammation. However, in several studies pro- static inflammation and calcification have been implicated in the pathogenesis of prostate cancer (CaP). We evaluated the prevalence of PC during transrectal ultrasound (TRUS) and correlate the ultrasonographic patterns with histological findings. Methods: A prospective study of 664 patients undergoing TRUS and prostate biopsy was planned. A standardized reproducible technique was used with using a GE Logiq 7 machine equipped with a 5-9MHz multi-frequency convex probe “end-fire”. We defined marked presence of PC as multiple hyperechoic foci with significant area (≥ 3 mm in the largest diameter). PC were classified according to zone distribution into the gland: transitional zone (TZ), central zone (CZ), and peripheral zone (PZ). Results: No significant difference was noted between the patients with PC and without PC, when comparing age, preoperative PSA level, prostate volume, and biopsy number, except for DRE findings. 168 patients (25.3%) had marked presence of PC on TRUS: 50.6% in TZ, 20.2% in CZ, and 29.2% in PZ. 31 patients (63.3%) with presence of PC in PZ had CaP on biopsy. The correlation observed between CaP and the presence of PC in PZ was statistically significant (p < 0.001). However, among patients in the CaP group there was no statistical association between PC and moderate or high Gleason grade. Conclusions: This study suggests that chronic prostatic inflammation and PC have a role in the biogenesis of cancer. CaP was more frequent in patients with PC in PZ of the gland, but was not associated with higher Gleason grade among these patients (p < 0.001). KEY WORDS: Prostatic calculi; Prostate cancer; Inflammation; Ultrasound; Risk factors. Submitted 15 November 2016; Accepted 15 December 2016 Summary No conflict of interest declared. with age (3, 4). The study of prostate with transrectal ultrasound (TRUS) provides the evaluation of the number, location, and dimension of the PC through axial and sagit- tal views. PC are usually accepted associated with benign prostatic hyperplasia (BPH) or chronic prostatitis (5). However, in several studies prostatic inflammation and calcification have been implicated in pathogenesis of prostate cancer (CaP) (6-8). In this study we wished to evaluate the prevalence of PC during TRUS and correlate the ultrasonographic patterns with histological findings. MATERIALS AND METHODS A single center prospective randomized study of 664 consecutive patients referred for transrectal ultrasound- guided prostate biopsy (TRUSBx) to our Department was performed between November 2013 to April 2016. All patients underwent an initial TRUSBx for abnormal digital rectal examination (DRE), high prostate-specific antigen (PSA) levels (≥ 4 ng/ml), or both. Patients with a history of biopsy, surgical treatment for prostatic disease or neoadjuvant therapy were excluded from our study. All patients enrolled in the study signed a consent form for the procedure. TRUSBx was performed with the patient in the left lateral decubitus using a General Electric Logiq 7 machine (GE Healthcare, Milwaukee, WI, USA) equipped with a 5-9MHz multi-frequency convex probe “end-fire”. The patients were treated under local anaesthesia with Lidocaine Spray 10 gr/100 ml (ECOCAIN®, Molteni Dental, FI, Italy) applied two min- utes before the procedure (9). Each TRUS performed included an assessment of the prostatic diameter, the vol- ume of the whole prostate, the transition zone, capsular, seminal vesicle characteristics, presence/absence of pro- static calcification, and a morphological description of potential pathological features. The prostate volume was invariably calculated using prostate ellipse formula (0.52 x length x width x height). We defined moderate/marked presence of PC as multiple (≥ 3 in number) hyperechoic foci with significant area (≥ 3 mm in the largest diameter) and coarse shadow detected in both dimensions (Figure 1). Mild calcifica- tions were defined as 1 or multiple small foci without DOI: 10.4081/aiua.2016.4.304 Presented at 20th National Congress SIEUN, Sciacca 2016 INTRODUCTION Prostatic calculi (PC) are usually ovoid bodies, with vari- able sizes and shapes, and they are found in alveoli of pro- static glands (1). PC are created as a result of deposition of calcareous calcium salts on corpora amylacea (2). Their incidence is believed to begin after puberty and increase Dell'Atti-Prostatic calculi detected_Stesura Seveso 09/01/17 10:04 Pagina 304 305Archivio Italiano di Urologia e Andrologia 2016; 88, 4 Relation between prostatic calculi and cancer coarse shadow. PC were classified according to zone dis- tribution into the gland [transitional zone (TZ), central zone (CZ), and peripheral zone (PZ)]. All measurements were analysed and recorded by an experienced urologist. After having images of the prostate, sampling was carried out with a 18-Gauge Tru-Cut (Bard Biopsy Systems, Tempe, AZ, USA) needle powered by an automatic spring-loaded biopsy disposable gun. A 14-core biopsy scheme was performed in each patient, as first intention, including 2 basal samples (lateral and medial), 2 parasagittal samples (lateral and medial), 2 apical sam- ples (lateral and medial), and 1 transitional zone sample on each side. All biopsy cores were analysed internally by our Pathology Department specializes in genitourinary pathology. The Gleason grading was based on the rec- ommendations of the 2005 international society of uro- logical pathology consensus conference. Statistical analysis Comparisons between patients with PC and without PC were performed using the Mann-Whitney U test for con- tinuous variables and the chi-square test or Fisher’s exact test for categorical variables. Univariate logistic regres- sion analysis was used to identify the individual clinical factors predictive of CaP presence. All statistical analyses were conducted on Microsoft Excel 2010 platform ver- sion 10.1. A p < 0.05 was considered to indicated statis- tical significance. RESULTS The mean ± standard deviation age of enrolled patients was 61.4 ± 6.6 years, with a prostate volume (PV) of 46.2 ± 18.9 ml, initial PSA levels of 7.5 ± 5.3 ng/ml. The num- ber of biopsy cores was 11.4 ± 4.6. No significant differ- ence was noted between the patients with PC and with- out PC, when comparing age, preoperative PSA level, prostate volume, and number of biopsy fragments, except for DRE findings. In patients with PC more fre- quently abnormal DRE findings were observed. CaP was detected in 213 patients (32.1%) and their Gleason scores were ≤ 6 (51.2%), 7 (32.4%), and ≥ 8 (16.4%) (Table 1). High-grade prostatic intraepithelial neoplasia (PIN) was shown in 81 patients (12.2%), and atypical small acinar proliferations (ASAP) in 55 patients (8.3%). One hundred and sixty eight patients (25.3%) had marked presence of PC on TRUS: 85 patients (50.6%) in TZ, 34 patients (20.2%) in CZ, and 49 (29.2%) in PZ (Table 2). Prostatic calculi in TZ were frequently associ- ated with histological findings of inflammation (55.3%; p < 0.002) and BPH (41.2%; p < 0.001). However, in 31 patients (63.3%) with presence of PC in PZ had CaP on biopsy (p < 0.001). The frequency of CaP increased as Table 1. Demographic, clinic-pathologic features and outcomes of patients undergoing transrectal ultrasound biopsy. Patients with Patients without P prostatic calculi prostatic calculi (n: 168) (n: 496) Age (year), mean ± SD* 61.1 ± 6.3 62.4 ± 6.5 NS** Prostate volume (ml), mean ± SD* 45.4 ± 17.7 47.2 ± 18.3 NS** PSA’’ level (ng/ml), mean ± SD* 6.9 ± 5.8 7.7 ± 5.1 NS** N° biopsy cores, mean ± SD* 11.2 ± 4.4 11.9 ± 4.6 NS** Abnormal DRE+, n (%) 91 (54) 156 (31) < 0.001 Prostate cancer, n (%) 45 (26.8) 168 (33.9) NS** Biopsy Gleason score, n (%) ≤ 6 31 (68.9) 78 (46.4) < 0.001 7 10 (22.2) 59 (35.1) < 0.001 ≥ 8 4 (8.9) 31 (18.5) < 0.001 SD* = standard deviation; PSA’’ = prostate-specific antigen; NS** = not significant; DRE+= digital rectal examination. Table 2. Distribution of prostatic calculi and histological findings in patients undergoing transrectal ultrasound biopsy. Prostatic calculi distribution Transitional Central Peripheral P Histological findings, zone zone zone n (%) (n: 85) (n: 34) (n: 49) Hyperplasia 35 (41.2) 10 (29.5) 10 (20.4) < 0.001 Inflammation 47 (55.3) 13 (38.2) 8 (16.3) < 0.002 Cancer 3 (3.5) 11 (32.3) 31 (63.3) < 0.001 Figure 1. Transrectal ultrasound in axial section of the prostate gland: middle (A) and apex (B) zone show marked calcifications with multiple hyperechoic foci, and significant area (≥ 3 mm) with coarse shadow. Dell'Atti-Prostatic calculi detected_Stesura Seveso 09/01/17 10:04 Pagina 305 Archivio Italiano di Urologia e Andrologia 2016; 88, 4 L. Dell’Atti, A.B Galosi, C. Ippolito 306 patient age increased. In addition, patients with CaP had higher PSA levels and smaller prostates than men with- out CaP (p < 0.001). The correlation observed between CaP and the presence of PC in PZ of the prostatic gland was statistically significant (p < 0.001). However, among patients in the CaP group there was no statistical associ- ation between PC and moderate or high Gleason grade. DISCUSSION In literature, no studies have systematically looked for PC in a general population. However, it is generally accepted that the incidence of PC increases with age (10). In one study of 612 TRUS, PC were seen in 47.2% of patients younger than 50 years old and in 86% of those older than 50 years. The authors showed that calcifications are asso- ciated with chronic inflammation (11). Geramoutsos et al. (12) screened 1374 men younger than 50 years old and found 101 cases of PC (7.4%). The patients with larger significant calcifications were much more likely to have chronic inflammation. A casual connection between chronic inflammation and carcinogenesis was supposed for the first time by Virchow in 1863 (13). Today it is accepted that almost 25% of all cancers are associated to chronic inflammatory diseases. Standard examples are gastric cancer occurring on a history of Helicobacter pylori infection or colon carcinoma after history of Crohn’s disease (14). In the last years, the casual link between inflammation and PCa has been investigated (15). However, the cause of chronic prostatic inflamma- tion as well as its presumed role in carcinogenesis remain unclear. Inflammation and PC are often histologically apparent in the examination of the CaP specimens from older men (4). Histologically, chronic inflammatory cell infiltrates are commonly observed in prostate specimens in the peripheral and transitional zone of the gland (7). Immune surveillance may clarify the association between the inflammation and CaP. The prostate gland is clearly an immunocompetent organ. Besides epithelial and stromal cells, the prostate also contains a small number of immunocompetent cells (lymphocytes, macrophages and granulocytes), which are collectively known as human prostate-associated lymphoid tissue (16). Vignozzi and Maggi demonstrated that prostate stromal cells can act as antigen-presenting cells, stimulating alloreactive CD4+ T cells to produce several inflammatory cytokines, chemokines and growth factors (including IL 8, IL 6 and bFGF) in response to a variety of inflammatory stimuli (17). The formative mechanism of PC is not well under- stood, but is usually observed more often in an inflam- mation of prostate gland than in the prostates of men with CaP. Prostatic stones are hypothesized to be calcifications of prostatic secretions, with a core of calcium apatite sur- rounded by concentric layers (18). Multiple different aeti- ological agents are thought to contribute to initiation of PC, including infections, bacterial biofilm production, dietary factors, physical trauma, hormonal changes, desquamation of prostatic epithelium with obstruction of intraprostatic ducts and urine reflux (3, 8, 19). Griffiths et al. (20) reported 63% correlation between PC and CaP on TRUS. Similar results were found by Hwang et al. (21). In their study the authors deduced that, although not sta- tistically significant, PC were more common in men with CaP and were associated with higher Gleason grade among these patients. Contrary to the aforementioned researches Woods et al. (8) showed that PC were less com- monly associated with CaP than with BPH. Epidemiological, genetic, molecular and rodent model studies have suggested an association between PC and CaP. Zhang et al. (22) further examined the relation between inflammation, PC and PCa in surgical speci- mens. Inflammation was more common in radical prosta- tectomy specimens. The limitation of this tissue source relates to the fact that these prostates were removed from patients with an established diagnosis of PCa, and their aggressiveness warranted surgery. However, in our study, inflammation was presented histologically in association with PC in 46.4% of patients who were diagnosed PCa on prostate biopsy. This proportion of positive findings for CaP in the biopsy specimens is relatively high compared with those in previous studies. To our knowledge, this is the first prospective study based on a large number of patients to show a link between CaP and PC. However, it had several limitations. A first limitation, we had no data available regarding the ethnic background of the patients. This details could be of special interest, because in multi- ethnic populations, some subgroups might have more unfavourable CaP characteristics than others (23). However, although we did not expressly documented race, the majority of the patients of our study cohort were white and Italian population. Thus, the number of Asian and black patients was very small and surely did not exceed 1% of the entire cohort. Second in this study only a visual assessment of PC was performed. The description of PC, calcification size, hyperechoic prostatic images as signs of an inflammation is not usually accepted in the urological community (24). We did not quantify the extent of PC and distinguished only 2 distinct types of severity. To clarify the actual role of PC on CaP develop- ment more molecular research, cytokines and inflamma- tory markers are required, and would provide a more accurate measure of inflammation. Finally, long term fol- low-up of patients with PC might clarify the possible association between PC, inflammation and CaP. There is no evidence confirming a temporal relationship between tissue inflammation and prostatic carcinogenesis. CONCLUSIONS Although we don’t completely understand the role of the PC in the development of BPH or CaP. There is emerging evidence that inflammation is crucial for the etiology of BPH, whether inflammation has a role in the pathogene- sis of CaP remains unclear. Chronic prostatic inflamma- tion may result from the immunologic response of differ- ent pathogenic causes that produce a tissue damage and subsequent vulnerability to developing cancer. However, further studies are necessary to more fully elucidate the relationship between inflammation, PC, and CaP. REFERENCES 1. Sfanos KS, Wilson BA, De Marzo AM, et al. Isaacs WB. Acute inflammatory proteins constitute the organic matrix of prostatic cor- Dell'Atti-Prostatic calculi detected_Stesura Seveso 09/01/17 10:04 Pagina 306 307Archivio Italiano di Urologia e Andrologia 2016; 88, 4 Relation between prostatic calculi and cancer pora amylacea and calculi in men with prostate cancer. Proc Natl Acad Sci. 2009; 106:3443-8. 2. Shoskes DA, Lee CT, Murphy D, Kefer J, et al. Incidence and sig- nificance of prostatic stones in men with chronic prostatitis/chronic pelvic pain syndrome. Urology. 2007;70:235-8. 3. Park SW, Nam JK, Lee SD, Chung MK. Are prostatic calculi inde- pendent predictive factors of lower urinary tract symptoms? Asian J Androl. 2010; 12:221-6. 4. Suh JH, Gardner JM, Kee KH, et al. Calcifications in prostate and ejaculatory system: a study on 298 consecutive whole mount sections of prostate from radical prostatectomy or cystoprostatectomy speci- mens. Ann Diagn Pathol. 2008; 12:165-70. 5. Kim WB, Doo SW, Yang WJ, et al. Influence of prostatic calculi on lower urinary tract symptoms in middle-aged men. Urology. 2011; 78:447-9. 6. Yun BH, Hwang EC, Yu HS, et al. Is histological prostate inflam- mation in an initial prostate biopsy a predictor of prostate cancer on repeat biopsy? Int Urol Nephrol. 2015; 47:1251-7. 7. Smolski M, Turo R, Whiteside S, et al. Prevalence of prostatic cal- cification subtypes and association with prostate cancer. Urology. 2015; 85:178-81. 8. Woods JE, Soh S, Wheeler TM. Distribution and significance of microcalcifications in the neoplastic and non neoplastic prostate. Arch Pathol Lab Med. 1998; 122:152-5. 9. Dell’Atti L. Lidocaine spray administration in transrectal ultra- sound-guided prostate biopsy: five years of experience. Arch Ital Urol Androl. 2014; 86:340-3. 10. Delongchamps NB, de la Roza G, Chandan V, et al. Evaluation of prostatitis in autopsied prostates--is chronic inflammation more associated with benign prostatic hyperplasia or cancer? J Urol. 2008; 179:1736-40. 11. Bock E, Calugi V, Stolfi V, et al. Calcifications of the prostate: a transrectal echographic study. Radiol Med. 1989; 77:501-3. 12. Geramoutsos I, Gyftopoulos K, Perimenis P, et al. Clinical cor- relation of prostatic lithiasis with chronic pelvic pain syndromes in young adults. Eur Urol. 2004; 45:333-7. 13. Woenckhaus J, Fenic I. Proliferative inflammatory atrophy: a background lesion of prostate cancer? Andrologia. 2008; 40:134-7. 14. Lu H, Ouyang W, Huang C. Inflammation, a key event in can- cer development. Mol Cancer Res. 2006; 4:221-33. 15. Sfanos KS, De Marzo AM. Prostate cancer and inflammation: the evidence. Histopathology. 2012; 60:199-215. 16. De Marzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate carcinogenesis. Nat Rev Cancer. 2007; 7:256-69. 17. Vignozzi L, Maggi M. Prostate cancer: intriguing data on inflammation and prostate cancer. Nat Rev Urol. 2014; 11:369-70. 18. Gu M, Li W, Chen Q, et al. Prostate calculi can higher urinary retention probability and worsen uncomfortable feeling after prostate biopsy but not predict cancer. Int J Clin Exp Med. 2015; 8:6282-6. 19. Irani J, Levillain P, Goujon JM, et al. Inflammation in benign prostatic hyperplasia: correlation with prostate specific antigen value. J Urol. 1997; 157:1301-3. 20. Griffiths GJ, Clements R, Jones DR, et al. The ultrasound appearances of prostatic cancer with histological correlation. Clin Radiol. 1987; 38:219-27. 21. Hwang EC, Choi HS, Im CM, et al. Prostate calculi in cancer and BPH in a cohort of Korean men: Presence of calculi did not cor- relate with cancer risk. Asian J Androl. 2010; 12:215-20. 22. Zhang W, Sesterhenn IA, Connelly RR, et al. Inflammatory infil- trate (prostatitis) in whole mounted radical prostatectomy specimens from black and white patients is not an etiology for racial difference in prostate specific antigen. J Urol. 2000; 163:131-6. 23. Ravery V, Dominique S, Hupertan V, et al. Prostate cancer char- acteristics in a multiracial community. Eur Urol. 2008; 53:533-538. 24. Ludwig M, Weidner W, Schroeder-Printzen I, et al. Transrectal prostatic sonography as a useful diagnostic means for patients with chronic prostatitis or prostatodynia. Br J Urol. 1994; 73:664-8. Correspondence Lucio Dell’Atti, MD, PhD (Corresponding Author) dellatti@hotmail.com Carmelo Ippolito, MD Department of Urology - University Hospital “St. Anna” 8 A. Moro Street, 44124 Cona, Ferrara, Italy Andrea B Galosi, MD Division of Urology, Marche Polytechnic University Ancona, Italy Dell'Atti-Prostatic calculi detected_Stesura Seveso 09/01/17 10:04 Pagina 307