Advances in Urological Diagnosis and Imaging - AUDI (Vol. 4 - n. 3 - 2021)

ISSN 2612-7601

ADVANCES IN UROLOGICAL DIAGNOSIS AND IMAGING

Vol. 4 - n. 3 - 2021

EDITOR IN CHIEF Andrea B. Galosi CO-EDITOR Pasquale Martino

SPECIAL ISSUE ON RENAL CANCERS

OFFICIAL JOURNAL of

S.I.E.U.N.

Italian Society of Integrated Diagnostic in Urology, Andrology, Nephrology

PRESENTANO

ATLANTE di ECOGRAFIA UROLOGICA, ANDROLOGICA e NEFROLOGICA a cura di

PASQUALE MARTINO

124 autori 592 pagine + di 1500 immagini ecografiche 61 video Hardcover Cofanetto

Costo di copertina € 180,00

Sconto del 50% valido fino al 31 gennaio 2022 (Indicando il Codice: INV21).

Prezzo finale: € 90,00* L’opera è acquisabile inviando un’e-mail a: atlantediecografia@gmail.com * IVA e Spese di spedizione comprese

ADVANCES

IN

UROLOGICAL DIAGNOSIS AND IMAGING

Official Journal of S.I.E.U.N. EDITOR in CHIEF Andrea B. Galosi, Ancona (IT)

CO-EDITOR Pasquale Martino, Bari (IT)

ASSISTANT EDITOR Lucio Dell’Atti, Ancona (IT)

ASSISTANT EDITOR JUNIOR Carlo Giulioni, Ancona (IT)

EDITORIAL BOARD Urology Ahmed Hashim, London (GB), Artibani Walter, Verona (IT) Battaglia Michele, Bari (IT), Bucci Stefano, Trieste (IT) Carini Marco, Firenze (IT), Carrieri Giuseppe, Foggia (IT) De Nunzio Cosimo, Roma (IT), Fandella Andrea, Treviso (IT) Ficarra Vincenzo, Messina (IT), Finazzi Agrò Enrico, Roma (IT) Franzese Corrado, Nola (IT), Gunelli Roberta, Forlì (IT) Kastner Christof, Cambridge (GB), Lapini Alberto, Firenze (IT) Miano Roberto, Roma (IT), Mirone Vincenzo, Napoli (IT) Montorsi Francesco, Milano (IT), Morgia Giuseppe, Catania (IT) Muller Stefan, Bonn (GE), Palazzo Silvano, Bari (IT) Pavlovich Christian, Baltimore, Maryland (USA) Pepe Pietro, Catania (IT), Rocco Bernardo, Modena (IT) Salomon George, Hamburg (GE) Schiavina Riccardo, Bologna (IT), Scattoni Vincenzo, Milano (IT) Volpe Alessandro, Novara (IT), Waltz Joachen, Marseille (FR)

Andrology Bettocchi Carlo, Bari (IT), Bitelli Marco, Roma (IT) Cai Tommaso, Trento (IT), Cormio Luigi, Foggia (IT) Fusco Ferdinando, Napoli (IT), Gontero Paolo, Torino (IT) Liguori Giovanni, Trieste (IT), Lotti Francesco, Firenze (IT) Pizzocaro Alessandro, Milano (IT), Trombetta Carlo, Trieste (IT)

Nephrology Boscutti Giuliano, Trieste (IT), D’Amelio Alessandro, Lecce (IT), Fiorini Fulvio, Rovigo (IT), Gesualdo Loreto, Bari (IT), Granata Antonio, Agrigento (IT), Ranghino Andrea, Ancona (IT)

Radiology Barozzi Libero, Bologna (IT), Bertolotto Michele, Trieste (IT) Giuseppetti Gian Marco, Ancona (IT), Giovagnoni Andrea, Ancona (IT), Valentino Massimo, Tolmezzo (IT)

Pathology Beltran Antonio Lopez, Lisbon (PT) Fiorentino Michelangelo, Bologna (IT) Liang Cheng, Indianapolis (USA), Montironi Rodolfo, Ancona (IT)

Bio-Medical Engineering Wijkstra Hessel, Eindhoven (NL)

Advances in Urological Diagnosis and Imaging - 2021; 4, 3

I

Official Journal of S.I.E.U.N.

General Information

Contents

AIMS and SCOPE

41

“Advances in Urological Diagnosis and Imaging” (AUDI) has the purpose of promoting, sharing and favorite technical-scientific research on echography and imaging diagnosis, in diagnostic and terapeutical field of Urology, Andrology and Nefrology. AUDI publishes original articles, reviews, case reports, position papers, guidelines, editorials, abstracts and meeting proceedings.

AUDI is Open Access at www.issuu.com Why Open Access? Because it shares science at your finger tips with no payment. It is a new approach to medical literature, offering accessible information to all readers, becoming a fundamental tool, improving innovation, efficiency and interaction among scientists.

Carlo Giulioni, Manuel Di Biase, Giuseppe Sortino, Willy Giannubilo,Vincenzo Ferrara

46

Use of renal scoring systems in nephron sparing surgery: applicability and reproducibility from residents’ perspective Carlo Giulioni, Simone Scarcella, Edoardo Agostini, Silvia Stramucci, Lucio Dell’Atti, Andrea Benedetto Galosi

51

Liquid biopsy in Renal Cell Carcinoma: Current and future applications

Alessia Cimadamore, Simone Scarcella, Erika Palagonia, Lucio Dell’Atti, Andrea Benedetto Galosi, Rodolfo Montironi

COPYRIGHT

Papers are accepted for publication with the understanding that no substantial part has been, or will be published elsewhere. By submitting a manuscript, the authors agree that the copyright is transferred to the publisher if and when the article is accepted for publication. The copyright covers the exclusive rights to reproduce and distribute the article, including reprints, photographic reproduction and translation.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the Publisher.

Oncological and functional outcomes of clampless retroperitoneoscopic Nephron-Sparing Surgery for intermediate to high complexity kidney tumors

55

A Diagnostic Challenging: a Retroperitoneal Mass

Andrea Benedetto Galosi, Simone Scarcella, Erika Palagonia, Daniele Castellani, Carlo Giulioni, Lucio Dell’Atti, Alessia Cimadamore

BUSINESS INFORMATION

Price for single printed issue: Euro 20,00 Annual subscription rate for the printed version (3 issues) is Euro 52,00.

Subscription orders should be sent to: Edizioni Scripta Manent s.n.c.

Via Melchiorre Gioia 41/A - 20124 Milano, Italy Tel. +39 0270608060 - e-mail: info@edizioniscriptamanent.eu Payments should be made by Bank transfer to: Edizioni Scripta Manent s.n.c.

Unicredit Milano IBAN: IT 23 K 02008 01749 000100472830 ADVERTISING For details on media opportunities within this journal please contact Mrs. Donatella Tedeschi, MD at +39 0270608060

Edizioni Scripta Manent s.n.c.

Via Melchiorre Gioia 41/A - 20124 Milano, Italy Tel. +39 0270608060 Registrazione: Tribunale di Milano n. 19 del 17/01/2018 e-mail: info@edizioniscriptamanent.eu web: www.edizioniscriptamanent.eu

Direttore Responsabile: Pietro Cazzola Direzione Marketing e PR: Donatella Tedeschi Comunicazione e Media: Ruben Cazzola Grafica e Impaginazione: Cinzia Levati Affari Legali: Avv. Loredana Talia (MI) Stampa: Àncora s.r.l. (MI)

The total or partial reproduction, by any means, is forbidden without the written authorization of the Publisher.

The Publisher is not liable for the opinion expressed by the Authors of the articles and for images used by them.

II

Advances in Urological Diagnosis and Imaging - 2021; 4, 3

ORIGINAL

PAPER

Oncological and functional outcomes of clampless retroperitoneoscopic Nephron-Sparing Surgery for intermediate to high complexity kidney tumors Carlo Giulioni 1, Manuel Di Biase ², Giuseppe Sortino ², Willy Giannubilo ², Vincenzo Ferrara ² 1 “Ospedali Riuniti” University Hospital, Department of Urology, Ancona 2 “Carlo Urbani” Hospital, Department of Urology, Jesi (Italy).

(Italy);

Introduction. Aim of this retrospective study is to evaluate oncological and functional outcomes in a consecutive series of patients submitted to retroperitoneal clampless tumor enucleation (RCTE) for intermediate and high complexity renal masses according to RENAL nephrometry score.

Material and methods. We evaluated patients treated with RCTE from January 2013 to January 2020, in a referral centre.

Inclusion criterium was renal masses with intermediate or high complexity according to the RENAL score.

Results. Overall 77 patients underwent RCTE median operative time was 125 min, median intraoperative blood loss was 225 ml.

In no cases renal artery clamping or conversion to radical nephrectomy was necessary. The median decrease in haemoglobin 24 h after surgery was 2 g/dL and the median length of stay was 5 days.

14 minor complications (Clavien-Dindo 1 or 2) and 3 major ones (Clavien-Dindo 3a or higher) were reported. MIC score was achieved in 71 (92%) cases. With a median follow up time of 27 months, no differences in renal function and oncological outcomes were recorded.

Conclusions. Our study showed that Retroperitoneoscopic NSS was associated with low operative time and intraoperative blood loss. Moreover, simple enucleation ensures the preservation of renal function with the maintenance of optimal oncologic radicality.

SUMMARY

KEY WORDS: Laparoscopy, tumor enucleation, clampless, kidney tumors.

INTRODUCTION The kidney tumor is among the most common cancers and accounts for 2.2% of cancer diagnoses globally (1).

In most cases, however, the finding is purely incidental through routine radiologic examinations, so the diagnosis occurs in the early stage. Therefore, if previously nephronsparing surgery (NSS) was used in cases of maximum need for preservation of healthy tissue, such as chronic kidney disease (CKD), solitary kidney or bilateral multifocal renal masses, currently the indication has expanded. Indeed, according to the updated guidelines, this procedure is also recommended in cases of clinical T1b tumors (2). However, in addition to tumor size, the further anatomical characteristics of the renal mass, such as its exophytic component or its relationship with the collector system, are crucial. In the last decades, several scoring systems were devised to assess the complexity of NSS, including the Radius Exophytic/Endophytic Nearness Anterior/Posterior Location (RENAL) nephrometry score (3).

Laparoscopic partial nephrectomy (LPN) was previously considered more difficult and time-consuming than open technique (4), while, nowadays, depending on the surgeon’s expertise, both open and minimally invasive (laparoscopic or robot-assisted) approaches are considered valid (2). Furthermore, Gill et al. reported that the laparoscopic approach results in lower rates of postoperative bleeding than the open one (5).

According to the standard partial nephrectomy, the renal artery is clamped before tumor resection to avoid massive intraoperative blood loss, although this this is a negative prognostic factor for long-term renal function. Indeed, warming ischemia time (WIT) is a significant predictor at 48 months after surgery and correlates with the amount of residual functional parenchyma (6). Currently, the application of the off-clamp technique is known for comparable perioperative safety, equivalent oncologic outcomes, and superior long-term renal function preservation compared to on-clamp (7).

Our study aims to evaluate the oncological and functional outcomes of retroperitoneoscopic clampless tumor enuAdvances in Urological Diagnosis and Imaging - 2021; 4,3

41

C. Giulioni, M. Di Biase, G. Sortino, W. Giannubilo, V. Ferrara

cleation (RCTE) for renal masses with a RENAL score of 7 or higher.

MATERIALS

AND METHODS

PATIENTS

Clinical data of 164 patients who underwent partial nephrectomy from January 2013 to January 2020 were retrospectively collected. The laparoscopic procedures have been performed by a single experienced surgeon, using the same standardized technique. All patients were previously evaluated by contrast-enhanced computed tomography (CT) scan of the abdomen and RENAL nephrometry score was calculated to estimate the surgical complexity of the masses. Patients with a renal mass with a low complexity were excluded. Therefore, demographic characteristics of 77 patients, including age, gender and preoperative data were recorded, as reported in Table 1.

Comorbid conditions were categorized through the Charlson comorbidity index and American Society of Anesthesiologists (ASA) physical status classification system score. Intraoperative records were collected, comprising operative time (OT), estimated blood loss (EBL), Warm Ischemia Time (WIT), eventual radical nephrectomy conversion rate. Every postoperative complication was recorded and ranked according to Clavien-Dindo Classification (8). Furthermore, we used the Margin, ischemia, and complications (MIC) score, defined by the absence of CD complications grade higher than 2, no positive surgical margins and ischemia time less than 20 minutes (9), to assess the NSS safety. Preoperative and 1-year postoperative eGFR (Pre- and 1YPO-eGFR), estimated with MDRD formula, were recorded and compared to renal function worsening.

SURGICAL TECHNIQUE

The procedure was performed using the 3-trocar technique. After the general anesthesia, the patient was positioned at the flank position. The skin was cut near the tip of the 12th rib and then two 10-mm trocars were positioned under digital guidance, one at the anterior axillary line and one at the posterior one. Then, the 12-mm trocar for the camera was placed.

After the dissection of perirenal fat and, subsequently, Gerota’s fascia, hilar vessels were identified, and, in case of T1b or endophytic masses, skeletonized. An ultrasonographic probe might be used for renal masses not visible at the renal surface. The mass was identified, and cold blade scissors were used for the tumor excision. During this step, bleeding from the dissection site is controlled by bipolar forceps. Once the tumor enucleation was completed, absorbable hemostats were applicable when further bleeding occurs. Afterwards, when necessary, parenchymal 2-0 sutures with sliding clip technique might be applied. Finally, the mass was removed through a 10mm retriever bag.

STATISTICAL ANALYSIS

Qualitative variables were described using absolute frequencies and percentages. Quantitative variables were

42

Advances in Urological Diagnosis and Imaging - 2021; 4,3

described using the median and interquartile range.

Follow-up changes in biomarkers were calculated and analyzed using a Wilcoxon Test. IBM SPSS (V26) was used as statistical software.

All statistical tests were two-tailed and p< 0.05 was considered to indicate statistical significance.

RESULTS Overall, 77 patients underwent RCTE for renal masses with intermediate/high complexity. As shown in Table 1, the median tumor size was 44 (40-54) mm, and the median RENAL score was 7 (7-8).

All perioperative data are summarized in Table 2. Median Operative Time (OT) was 125 (100-160) min and median Estimated Blood Loss (EBL) was 225 (170-310) ml. All the procedures did not require renal artery clamping. The median length of stay was 5 (4-6) days, while the median 24h-decrease in Hb was 2 (1,1-2,8) g/dL. Radical nephrectomy was not necessary in any cases. At least one postoperative complication occurred in 17 patients: fourteen of them were minor, while there were 3 cases of major com-

Table 1. Descriptive characteristics of patients in the RCTE group. Data are presented as medians (interquartile range) and frequencies (proportions).

Variables

LPN (n= 77)

Age, years

65 (57-72)

BMI, kg/m2

26,2 (24,3-28,7)

Sex Male Female

49 (64) 28 (36)

Charlson Comorbidity Index

5 (3-6)

ASA Score

2 (2-3)

Tumor Side Right Left

37 (48) 40 (52)

Tumor size, mm

44 (40-54)

T stage T1a T1b T2

17 (22) 44 (57) 16 (21)

Tumor depth Completely exophytic Partial exophytic Complete endophytic

11 (14) 47 (61) 19 (25)

RENAL score

7 (7-8)

RENAL score stage Intermediate High

70 (91) 7 (9)

Preoperative Creatinine Preoperative eGFR

0,9 (0,73-1,09) 91,5 (70,3-119,2)

Oncological and functional outcomes of clampless retroperitoneoscopic Nephron-Sparing Surgery for intermediate to high complexity kidney tumors

plications (CD 3a or more). One patient required admission to the intensive care unit (ICU) due to atrial fibrillation, one required re-surgery for haemostasis, and the other underwent Super-Selective Embolization for a renal pseudoaneurysm. More details about postoperative complications are reported in Table 3. Based on its criteria, the MIC score was achieved in 71 (92%) cases.

Median follow-up (FU) was 27 (14-63) months. At routinely FU controls no cases of relapse were detected in all

Table 2. Perioperative data of patients related to the RCTE group. Data are presented as medians (interquartile range) and frequencies (proportions).

WIT: Warm Ischemia Time; CD: Clavien-Dindo; MIC: Margin Ischemia Complications; 1MPO: 1-Month Postoperative; 1YPO: 1-Year Postoperative.

Variables

LPN (n= 77)

Operative time, min

125 (100-160)

Estimated blood loss, mL

225 (170-310) 0

WIT, min Conversion to radical nephrectomy Yes No

0 (0) 77 (100)

24h-decrease in Hemoglobin, g/dL

2 (1,1-2,8)

Length of Stay, days

5 (4-6)

At least one postoperative complication Yes No

17 (22) 45 (78)

groups. The overall survival rate at 5 years was 100%.

Furthermore, in terms of functional outcome, no significant difference was observed between Pre- and 1-YPO eGFR (p=0,15), as reported in Table 4.

DISCUSSION Small renal masses were historically treated with partial nephrectomy due to its oncologic radicality and preservation of long-term renal function compared to radical nephrectomy, but, currently, this technique is recommended even for higher complexity tumors through the surgeon’s skill set and available resources. (10). In multicenter observational studies, both open and minimally invasive approaches have shown satisfactory functional and oncological results for greater tumors, with comparable postoperative complications and oncological outcomes (11, 12).

Moreover, in addition to the partial nephrectomy, several resection techniques were devised to maximize the preservation of healthy parenchyma, as firstly wedge resection, which includes a small part of surrounding tissue, and then tumor enucleation (TE), with the removal solely of the mass. Although the latter is considered dangerous for local recurrences due to no ablation of the tumor bed, this procedure showed similar oncological outcomes compared to partial nephrectomy in a comparative

Table 3. Postoperative complications of patients related to the RCTE group. Data are presented as frequencies (proportions).

Complication

CD classification CD1 CD2 CD3a or more

8 (10) 6 (8) 3 (4)

Positive Surgical Margin Yes No

3 (4) 74 (96)

MIC Score Yes No

71 (92) 6 (8)

1MPO Creatinine

0,94 (0,77-1,25)

1MPO eGFR

87,9 (60,7-1111)

1YPO Creatinine

0,95 (0,77-1,14)

1YPO eGFR

89,0 (67,0-110,7)

LPN (n= 77)

Clavien-Dindo 1 Fever Pleural injury Postoperative anemia Pancreatic injury Spleen injury

8 (9) 3 (4) 2 (4) 2 (2) 0 (0) 0 (0)

Clavien-Dindo 2 Pneumonia Blood transfusion Acute kidney injury

6 (8) 1 (1) 3 (4) 2 (3)

Clavien-Dindo 3a or more Super-selective embolization Admission to ICU Urinary Leak

3 (4) 1 (1) 1 (1) 1 (1)

Table 4. Preoperative, 1MPO and 1YPO Renal Function compared using paired Wilcoxon test. Data are presented as medians (interquartile range) and proportions. Cr: Creatinine; Pre: Preoperative; 1MPO: 1-Month Postoperative; 1YPO: 1-Year Postoperative.

Variable

Pre value

1MPO value

Pre-1MPO p value

1YPO value

Pre-1YPO p value

Serum Creatinine, mg/dL

0,9 (0,73-1,09)

0,94 (0,77-1,25)

0,2

0,95 (0,77-1,14)

0,09

91,5 (70,3-119,2)

87,9 (60,7-1111)

0,1

89,0 (67,0-110,7)

0,15

eGFR, mL/min

Advances in Urological Diagnosis and Imaging - 2021; 4,3

43

C. Giulioni, M. Di Biase, G. Sortino, W. Giannubilo, V. Ferrara

study (13). Besides, Simone et al., evaluating the microscopic details of definitive surgical margin evaluation, reported that the presence of a thin layer of tissue allows for negative margins when tumor enucleation is performed (14). In the present study, 3 (4%) positive surgical margins have been detected. This result is consistent with a large case series, which attributed an acceptable range (0-4%) for laparoscopic partial nephrectomy (15). Thus, our study confirmed that tumor enucleation warrants optimal oncological outcomes. During follow-up, postoperative imaging did not report any relapse cases. However, careful and long-term surveillance for local recurrence is mandatory, particularly for RCC patients with positive surgical margins.

Laparoscopic NSS may be performed through a transperitoneal or retroperitoneal approach, depending on the location of the renal mass. The first approach is ideal for the bigger working space and freedom of movement with laparoscopic instruments (16), although bowel mobilization may determine alterations of global gastrointestinal motility and identification of the hilar vessels may be more difficult. Despite the spatial limitations and lack of view, the retroperitoneal approach allows easier access to certain tumors and major renal blood vessels, while the surgeon’s expertise ensures the orientation and identification of anatomical landmarks. However, according to Marszalek et al., transperitoneal laparoscopic and retroperitoneoscopic partial nephrectomy showed a similar overall complication rate, although the latter guarantees a lower operative time and length of hospital stay (17). In our results, no complications related to the bowel and other nearby organs, as the spleen or pancreas, occurred, confirming the retroperitoneal NSS safety.

During the NSS, WIT plays a key role in terms of functional outcomes. Indeed, it has a linear correlation with acute renal failure (ARF) and with the onset of new-onset stage IV CKD (18). However, its safe duration remains controversial. In the last decade, Trifecta (19) and MIC (9) were introduced to evaluate the quality of surgery and, respectively, 25 and 20 minutes are their upper bound. Moreover, according to Simmons et al., there is a positive association between tumor size and duration of renal artery clamping (20). In our study, zero-technique was feasible in all cases, regardless of renal mass size or complexity, and a nonsignificant loss of eGFR after NSS and at 1-year follow-up was reported. Therefore, we demonstrated that the clampless technique, aided by minimal excision of healthy renal parenchyma, provided optimal preservation of renal function. Besides, avoiding the renal artery clamping does not result in massive intraoperative haemorrhage and a worse view of the operative field during tumor enucleation (21).

In fact, in our case series involving intermediate/high renal masses, there was a low estimated median blood loss (225 mL), and blood transfusions were required in a few cases (3.9%), confirming the safety of RCTE. Moreover, soft coagulation can assist in achieving satisfying hemostasis.

Several matrix-thrombin sealants, porcine- or bovinederived gelatins, were developed. According to a recent meta-analysis, these hemostatic agents determine significant reductions in warm ischemia time, operative time,

44

Advances in Urological Diagnosis and Imaging - 2021; 4,3

and estimated blood loss, with comparable complications when used alone (22).

The limitations of the present study are evident. First, its retrospective nature. Second, we reported the outcomes of a small sample size without a comparison. Finally, all procedures were performed by an experienced laparoscopic surgeon and our results need to be confirmed also in centers with less skilled surgeons.

CONCLUSIONS In our series, RCTE has proven to be a safe and effective procedure even for renal masses with a RENAL score of 7 or higher; the retroperitoneoscopic approach allows direct access to the kidney and hilar vessels reducing operative time, while the clampless technique does not increase intraoperative blood loss and complication rates.

Excellent functional results were achieved, minimizing resection of healthy parenchyma ensuring optimal oncologic radicality. However, RCTE is advised to qualified surgeons with extensive laparoscopic experience.

REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6):394424.

2. B. Ljungberg (Chair), L. Albiges, J. Bedke, et al. Volpe Guidelines on Prostate Cancer. Edn. presented at the EAU Annual Congress Milan 2021. 978-94-92671-13-4. Publisher: EAU Guidelines Office. Place published: Arnhem, The Netherlands.

3. Kutikov A, Uzzo RG. The R.E.N.A.L. Nephrometry score: A comprehensive standardized system for quantitating renal tumor size, location and depth. J Urol. 2009; 182:844-53.

4. Gill IS, Desai MM, Kaouk JH, et al. Laparoscopic partial nephrectomy for renal tumor: duplicating open surgical techniques . J Urol 2002; 167:469-77 5. Gill IS, Kavoussi LR, Lane BR, et al. Comparison of 1,800 laparoscopic and open partial nephrectomies for single renal tumors. J Urol.

2007; 178(1):41-6.

6. Volpe A, Blute ML, Ficarra V, et al. Renal Ischemia and Function After Partial Nephrectomy: A Collaborative Review of the Literature.

Eur Urol. 2015; 68(1):61-74.

7. Liu W, Li Y, Chen M, et al. Off-clamp versus complete hilar control partial nephrectomy for renal cell carcinoma: a systematic review and meta-analysis. J Endourol. 2014; 28(5):567-76.

8. Clavien PA, Barkun J, de Oliveira ML, et al. The Clavien-Dindo classification of surgical complications: five-year experience. Ann Surg 2009; 250(2):187-196.

9. Buffi N, Lista G, Larcher A, et al. Margin, ischemia, and complications (MIC) score in partial nephrectomy: a new system for evaluating achievement of optimal outcomes in nephron-sparing surgery.

Eur Urol. 2012; 62(4):617-8.

10. Simmons MN, Weight CJ, Gill IS. Laparoscopic radical versus partial nephrectomy for tumors >4 cm: intermediate-term oncologic and functional outcomes. Urology. 2009; 73(5):1077-82.

11. Mari A, Tellini R, Porpiglia F, et al. Perioperative and Mid-term Oncological and Functional Outcomes After Partial Nephrectomy for Complex (PADUA Score ≥10) Renal Tumors: A Prospective

Oncological and functional outcomes of clampless retroperitoneoscopic Nephron-Sparing Surgery for intermediate to high complexity kidney tumors Multicenter Observational Study (the RECORD2 Project). Eur Urol Focus. 2020; S2405-4569(20)30210-8.

son of transperitoneal and retroperitoneal approaches. J Urol. 2005; 174:841-845.

12. Porpiglia F, Mari A, Bertolo R, et al. Partial Nephrectomy in Clinical T1b Renal Tumors: Multicenter Comparative Study of Open, Laparoscopic and Robot-assisted Approach (the RECORd Project).

Urology. 2016; 89:45-51.

17. Marszalek M, Chromecki T, Al-Ali BM, et al. Laparoscopic partial nephrectomy: a matched-pair comparison of the transperitoneal versus the retroperitoneal approach. Urology. 2011; 77(1):109-13.

13. Minervini A, Ficarra V, Rocco F, et al .Simple enucleation is equivalent to traditional partial nephrectomy for renal cell carcinoma: results of a nonrandomized, retrospective, comparative study. J Urol 2011; 185:1604-10.

14. Simone G, Papalia R, Guaglianone S, Gallucci M. “Zero ischaemia” sutureless laparoscopic partial nephrectomy for renal tumours with a low nephrometry score. BJU Int. 2012; 110(1):124-30.

15. Marszalek M, Carini M, Chlosta P, et al. Positive surgical margins after nephron-sparing surgery. Eur Urol. 2012; 61(4):757-63.

16. Wright JL, Porter JR. Laparoscopic partial nephrectomy: compari-

18. Thompson RH, Lane BR, Lohse CM, et al. Comparison of warm ischemia versus no ischemia during partial nephrectomy on a solitary kidney. Eur Urol. 2010; 58:331-6.

19. Zargar H, Allaf ME, Bhayani S, et al. Trifecta and optimal perioperative outcomes of robotic and laparoscopic partial nephrectomy in surgical treatment of small renal masses: a multi-institutional study.

BJU Int. 2015; 116:407-414.

20. Simmons MN, Schreiber MJ, Gill IS. Surgical renal ischemia: a contemporary overview. J Urol 2008; 180:19-30.

21. Weizer AZ, Gilbert SM, Roberts WW, et al. Tailoring technique of laparoscopic partial nephrectomy to tumor characteristics. J Urol.

2008; 180:1273-8.

CORRESPONDENCE Carlo Giulioni Department of Urology, University Hospital “Ospedali Riuniti”.

71 Conca Street, 60126, Ancona – Italy Email: carlo.giulioni9@gmail.com Phone: +39 320/7011978 ORCID: 0000-0001-9934-4011

Advances in Urological Diagnosis and Imaging - 2021; 4,3

45

ORIGINAL

PAPER

Use of renal scoring systems in nephron sparing surgery: applicability and reproducibility from residents’ perspective Carlo Giulioni, Simone Scarcella, Edoardo Agostini, Silvia Stramucci, Lucio Dell’Atti, Andrea Benedetto Galosi “Ospedali Riuniti” University Hospital, Department of Urology, Polytechnic University of Marche, Ancona (Italy).

Introduction. Aim of this retrospective study is to evaluate the reproducibility of four different nephrometry score systems (R.E.N.A.L., P.A.D.U.A., C-Index and ABC) comparing the values assigned by radiology and urology residents.

Material and Methods. From November 2019 to April 2021, we collected preoperative radiological data of 100 patients with a suspected diagnosis of kidney tumor. Three urologists and three radiologists, respectively at the first, second and last year of residency training program, evaluated images from CT scans and assessed the masses according to each nephrometry score.

Results. Intra-class correlation coefficient among all readers resulted higher for the RENAL and PADUA scores while it was lower for the C-Index and ABC score. Mean RENAL and C-Index scores were higher among urologists, while no difference was detected for both PADUA and ABC assessments. PADUA system resulted the only scoring system allowing comparable results for all residents, independently from the specific year of training. On the contrary, differences were detected for RENAL (comparing values between residents of the 1st and 2nd year, respectively), C-Index (between the 2nd and 5th year) and ABC (between residences of the 2nd year) scoring systems.

Conclusion. Nephrometry score based on anatomical characteristics, such as PADUA and RENAL, demonstrated a better correlation between urology and radiology residents. These scoring systems are reproducible, easy to use event between specialists with different training backgrounds and should routinely be included in the preoperative radiological evaluation by radiologists.

SUMMARY

KEY WORDS: Renal cell carcinoma, partial nephrectomy, renal score, residency program.

INTRODUCTION Renal cell carcinomas (RCCs) accounts for 2.2% of global cancer diagnoses and it is the seventh most common neoplasm diagnosed in developed countries (1). The incidence of newly diagnosed renal masses increased rapidly over the past decades, doubling its rate in Europe since 1975 (2).

The widespread adoption of cross-sectional radiological abdominal imaging techniques such as high resolution computed tomography (CT) and magnetic resonance imaging (MRI) determined a further increase in detection rates of incidental renal lesions smaller than 4 cm (3, 4).

Early diagnosis and surgical techniques advances led to a shift from radical treatment, which was considered the oncologic gold standard in the past, to a conservative approach with expanding indications for nephron sparing surgery (NSS) and cryoablation. In this setting, preoperative radiological imaging studies are of paramount importance to guide surgeons planning the best treatment option and surgical approach, according to the specific

46

Advances in Urological Diagnosis and Imaging - 2021; 4,3

anatomical characteristics of the renal masses, such as size, location and proximity to the urinary collecting system (8).

Several scoring systems (SS) have been developed to stratify patients and predict surgical complexity during tumor enucleation (9).

In 2009 Kutikov et Uzzo developed The RENAL nephrometry score (10) and during the same year Ficarra et al. published the PADUA classification system (11). Both scoring systems are based on the anatomical characterisation of renal masses concerning the following parameters: maximal tumour diameter, endophytic/exophytic growth ratio, anterior/posterior localization, relation with the kidney’s polar lines and proximity to renal sinus and urinary collecting system.

In 2010 Simmons et al. (12) presented the centrality index (C-index), a score system based on rigorous measurements of the distance between the maximum tumour diameter plane and the kidney hilar centre. Six years later, Spalviero et al. (13) validated the ABC scoring system taking into consideration the anatomical relationship between the

Use of renal scoring systems in nephron sparing surgery: applicability and reproducibility from residents’ perspective

tumour endophytic component, the arterial vascular anatomy of the kidney and the specific order of arterial branches needed to be dissected during NSS. These scores, representing the first generation of nephrometry scoring systems, are the most know and widely used ones. Different Authors, recently, developed and published the updated second generation of nephrometry scoring systems, that unfortunately has not been yet externally validated and extensively adopted in daily practice (14). However, despite the promising results of individual scoring systems, conflicting reports have been divulgated regarding the superiority of one score over the others (15). Due to the lack of a reference standard scoring system, the adoption and interpretation of SS may vary according to single centre’s preferences and physicians’ different training and levels of expertise. This may mislead preoperative radiological and surgical assessments, making data comparison difficult among surgical series. Although in the CT-radiology report the scoring system is not yet described, almost all current literature is based on renal scoring system to evaluate selection criteria to conservative surgery and surgical outcomes.

Considering the emerging role of residents’ in clinical and scientific activities in academic and non-academic hospitals, we observed a key-role of residents in Urology to assign tumor renal score other than to collect all medical records at hospital admission.

We aim to evaluate the applicability of four different nephrometry scoring systems in a real-life scenario, and to analyse their reproducibility by comparing the scores assigned by radiology and urology residents with different levels of expertise.

PATIENTS

AND METHODS

We enclosed prospectively 100 patients with CT showing a renal mass. All patients were candidate to nephron sparing surgery with a laparoscopic or open approach between November 2019 and April 2021 at our Urology Institute. All diagnostic Computed Tomography (CT) scans with contrast enhanced were electronically stored.

All images were retrospectively evaluated in the axial and coronal planes. Six residents in total, three urologists and three radiologists, respectively at the first, third and last year of residency training program, received a specific training regarding the application of four different Scoring Systems (SS) by referenced tutors from the specific department of employment (Urology or Radiology). Once proficient in SS assignments, they, independently, analysed the radiological scans of each patient and assigned the four different nephrometry scores.

The included scores were the R.E.N.A.L. (radius, exophytic/endophytic properties, nearness of tumor to the collecting system or sinus in millimetres, anterior/posterior location relative to polar lines), the P.A.D.U.A. (preoperative aspects and dimensions used for anatomic classification), the C–Index (centrality-index) and the ABC (arterial based complexity). In descriptive analysis all continuous variables were summarized with median and interquartile range, while categorical variables with absolute and rela-

tive frequencies. The scores given by the “Urologists group” and the “Radiologists group” were compared using the Mann-Whitney test and graphically with the Box Plot.

Intra-class correlation coefficient (ICC) was used for continuous scores reliability, while categorical variables were tested with the Cohen’s kappa (k). Inter-Observer reliability was calculated, first comparing the scores among all readers, then among the same specialty group and finally between urologists and radiologists according to the specific year of residency training program. Moreover, we compared the mean differences between the scores assigned by the two groups of urologists and radiologists.

Statistical data analysis was performed with IBM SPSS Statistics 20.

RESULTS ICC among all readers resulted higher for RENAL and PADUA scores while it was lower for C-Index and ABC scores. Considering the specific professional background, Urologist SS were characterized by an intra-class correlation index higher than 0.8, while in the Radiologist group only Renal and PADUA scores presented comparable results. On the contrary, both C-Index and ABC score were characterised by lower ICC results as depicted in Table 1.

The median difference in scores assigned between the two groups of residents (Urology Vs Radiology) resulted in 1 point and 0.6 point for RENAL score (p=0.012) and the C-Index (p<0.001) respectively, while no differences were observed in both PADUA and ABC scores (p>0.05) (Table 2 and Figure 1).

The Cohen’s Kappa values indicated a moderate agreement for R.E.N.A.L., PADUA and ABC score between radiologists and urologists at the 1st year while for Renal and ABC scores it resulted higher between residents at the fifth year (Table 3).

Low correlation was found between urologists and radiologists at the second year of residency training program, for all the scores. Using R.E.N.A.L. score, a significative difference was reported between radiologists and urologists at the first (P=0.03) and second (P=0.001) year of residency training program, while for C-Index a discrepancy between radiologists and urologists was statistically significative between residents at the second (P=0.004) and the fifth (P=0.001) years of residency training programs.

Only between the second year residents for ABC score, a statistically significative difference (P=0.001) was detected.

PADUA was the only nephrometry score with comparable results between residents, independently from the specific year of residency training program (Table 4).

DISCUSSION Preoperative planning in patients with renal cortical neoplasms relies on anatomical tumour characteristics and the use of SS allows the characterization of important variables such as tumour size, locations and nearness of vascular structures, in a standardized manner. Moreover, SS accurately reflect the surgical complexity of kidney lesions Advances in Urological Diagnosis and Imaging - 2021; 4,3

47

C. Giulioni, S. Scarcella, E. Agostini, S. Stramucci, D. Castellani, L. Dell’Atti, A. Galosi Table 1.

Intraclass correlation and Cohen’s Kappa (95% confidence interval) amongst all readers.

Table 2.

Comparison between urologists and radiologists median score system assessments.

Table 3. Cohen’s Kappa: comparison between urologists and radiologists according to year of residency training program.

R.E.N.A.L.

All readers Urologists Radiologists

PADUA

C-Index

ABC

ICC (95% CI)

ICC (95% CI)

ICC (95% CI)

ICC (95% CI)

0.80 (0.45 – 0.78)

0.82 (0.74 – 0.90)

0.40 (0.24 - 0.55)

0.52 (0.42 – 0.62)

0.91 (0.86 – 0.96)

0.86 (0.78 – 0.93)

0.81 (0.71 - 0.90)

0.81 (0.71 – 0.91)

0.80 (0.70 – 0.90)

0.83 (0.74 – 0.91)

0.46 (0.26 - 0.66)

0.32 (0.21 – 0.43)

R.E.N.A.L.

PADUA

C-Index

ABC

Median (IQR)

Median (IQR)

Median (IQR)

Median (IQR)

8 (7 – 9)

8 (7 – 10)

1.8 (1.3 – 2.6)

2 (2 – 3s)

7 (6 – 9)

8 (7 – 10)

2.4 (1.8 – 3.3)

2 (2 – 3s)

(Mann-Withney Test)

0.012

0.560

0.001

0.612

Residency Training Program Year

Kappa (95% CI)

Kappa (95% CI)

Kappa (95% CI)

Kappa (95% CI)

0.42 (0.36 – 0.48)

0.36 (0.28 – 0.44)

0.09 (0.07 – 0.11)

0.54 (0.44 – 0.64)

0.06 (0 – 0.12)

0.27 (0.20 – 0.34)

0.1 (0 – 0.2)

0.38 (0.28 – 0.48)

0.48 (0.42 – 0.54)

0.58 (0.50 – 0.66)

0.1 (0 – 0.2)

0.60 (0.41 – 0.69)

Urologists Radiologists P-value

Fifth Third First

R.E.N.A.L.

PADUA

C-Index

ABC

Table 4. Median and Interquartile Range (IQR): comparison between urologists and radiologists according to residency training program year.

R.E.N.A.L.

Median (IQR)

Urologists 5th Radiologists 5th Urologists 3th Radiologists 3th Urologists 1th Radiologists 1th

P

Wilcoxon test

7.5(6.5 – 9) 8(7 – 9)

0.06 0.001

0.55

8(7 – 9.5)

0.03

Advances in Urological Diagnosis and Imaging - 2021; 4,3

8(7 – 9)

P

Wilcoxon test

1.6(1.2 – 2.5)

0.20

2.45(1.9 – 3.25)

0.001

2.39(1.7 – 3.55)

P

Wilcoxon test

1(1-2)

0.77

1(1-2) 0.004

2.45(1.6 – 3.3) 0.76

ABC

Median (IQR)

1(1-2)

2.2(1.4 – 2.8)

8(7 – 10)

Figure 1. Box plot showing mean and ranges of scoring system by urologist and radiologist.

48

9(7 – 10)

C-Index

Median (IQR)

1.75(1.25 – 1.35)

8(7 – 9.5)

7(5 - 8) 7(6 – 9)

P

Wilcoxon test

8.5(7 – 10)

7.5(6 - 9) 7(6 – 9)

PADUA

Median (IQR)

1.5(1- 2)

0.001

1(0-1) 0.07

2(1-2)

0.05

Use of renal scoring systems in nephron sparing surgery: applicability and reproducibility from residents’ perspective

and guide the surgeons during the decision making process between RN and NSS. This is of paramount importance nowadays considering the expanding indications of nephron sparing surgeries. In the past nephron sparing techniques were reserved only for T1a stage renal masses, while recently we witnessed an incremental use of NSS even for stage T1b or greater tumors (17, 18). A preoperative radiological evaluation and standardised report of tumour characteristics is mandatory.

The use of these Scoring Systems should not be complex in its application and has to be based on objective measurements in order to represent the technical complexity of the planned surgery. All scores represent a standardized method for quantitatively reporting and categorizing renal tumors and can guide clinicians in choosing among different surgical approaches. Therefore, an ideal SS should be reproducible, in spite of the examiner's experience, and anticipate intra- and postoperative results. Veccia et al. (19) reported that RENAL and PADUA are the most feasible scores and the best to report surgical complexity and morbidity prediction. Bylund et al. (20) reported high complexity masses (based on RENAL, PADUA, C-Index scores) were positively associated with Warm Ischemia Time (WIT), which is considered a reliable surrogate of tumor complexity (21). Overall, these three classification systems performed better than tumor size and exophytic classification alone. Okhunov et al. (11) analyzed the relationship of the three scoring systems with various perioperative and postoperative variables. Interobserver reliability was excellent in all three scoring systems, showing interobserver correlations of 0.84 for the C-Index, 0.81 for the PADUA score, and 0.92 for the RENAL score.

All of the three systems showed a significant association with WIT and percentage change in creatinine level, while only C-Index did show a significant relationship for patients with lower scores having longer hospital stay.

In the present study, four nephrometry scores were compared through urology and radiology residents’ evaluation.

Our results showed that Scoring Systems based on anatomical characteristics are more reproducible: P.A.D.U.A. and R.E.N.A.L. score had the higher reproducibility among all groups. Only the first one showed comparable results for all years residents. Moreover, in the urologist group only there was a significant higher reproducibility of all the evaluated scores and we firmly believe that this finding is related to the daily-practice use of these SS compared to the radiologist group. On the other hand C-Index score tends to differ significantly between urologist and radiologist, even inside the same specialty. Sharma et al. (22) confirmed excellent concordance among specialists in scoring tumor characteristics through R.E.N.A.L. and P.A.D.U.A. C-index showed lower reproducibility among overall urologists and radiologists, probably due to difficult mathematical calculation.

Recently, different Authors investigated the correlation between different nephrometry scoring systems assigned by different specialists (23, 24). Monn et al. reported a better inter-reader correlation in scores assigned by radiologists in comparison to those assigned by urologists, with a mean two points increased score attributed by Urologists to complex renal masses (25). Moreover, Benadiba et al.

described a good reproducibility between nephrometry scoring systems assigned by urologists and radiologists, with a proportional significant association between high scoring values and increased incidence of major complications (23, 26).

To our knowledge only another study, within the scientific literature, evaluated the reproducibility of Scoring Systems, including a direct comparison between residents from urology and radiology. The Authors (25), compared PADUA and RENAL scores calculated by urology and radiology residents, finding a more reliable inter-reader reproducibility reliability using the PADUA scoring system (κ=0.40 vs κ=0.56). Moreover, the authors reported a higher prediction of conversion rates to radical nephrectomy using the PADUA scoring system. This is of outmost importance, considering that in the daily clinical practice radiological residents are more exposed to radiological imaging evaluation while the urological ones are directly involved in surgical planning and clinical assistance to patients (26).

Our study is not devoid from limitations. First, the size of the cohort was relatively small and larger number of patients are needed to verify our results. Second, all scores were assigned by trained residents with no revision by Consultant Urologists or Radiologists. Third, our work focused only on the assessment of nephrometry score and did not explore the correlation between different scores and peri-postoperative outcomes.

CONCLUSIONS Our results suggest that SS scores, assigned by urology and radiology residents, based on anatomical characteristics tend to show a higher reproducibility even among medical doctor in training with different professional backgrounds.

Renal scoring system should be included in urological and radiological scientific education and training programs.

Finally, considering that R.E.N.A.L. and P.A.D.U.A. scores showed the best accordance between groups, their use should be encouraged also by radiologists in the pre-operative report.

REFERENCES 1. Sanchez A, Feldman AS, Hakimi AA. Current Management of Small Renal Masses, Including Patient Selection, Renal Tumor Biopsy, Active Surveillance, and Thermal Ablation. J Clin Oncol. 2018; 36(36):35913600.

2. Epidemiology of Renal Cell Carcinoma, GLOBOCAN. 2020 3. Kaur R, Juneja M, Mandal AK. An overview of non-invasive imaging modalities for diagnosis of solid and cystic renal lesions. Med Biol Eng Comput. 2020; 58(1):1-24.

4. Campbell SC, Novick AC, Belldegrun A, et al. Guideline for management of the clinical T1 renal mass. J Urol. 2009; 182:1271-1279.

5. Van Poppel H, et al. A prospective, randomised EORTC intergroup phase 3 study comparing the oncologic outcome of elective nephron-sparing surgery and radical nephrectomy for low-stage renal cell carcinoma. Eur Urol. 2011; 59: 543.

6. Scosyrev E, et al. Renal function after nephron-sparing surgery verAdvances in Urological Diagnosis and Imaging - 2021; 4,3

49

C. Giulioni, S. Scarcella, E. Agostini, S. Stramucci, D. Castellani, L. Dell’Atti, A. Galosi sus radical nephrectomy: results from EORTC randomized trial 30904. Eur Urol. 2014; 65: 372.

7. Hora M, Eret V, Trávníček I, et al. Surgical treatment of kidney tumors - Contemporary trends in clinical practice. Cent Eur J Urol.

2016; 69(4):341-346.

8. Porpiglia F, Fiori C, Bertolo R, Scarpa RM. Does tumour size really affect the safety of laparoscopic partial nephrectomy? BJU Int 2011; 108:268-273.

9. Okhunov Z, Rais-Bahrami S, George AK, et al. The Comparison of Three Renal Tumor Scoring Systems: C-Index, P.A.D.U.A., and R.E.N.A.L. Nephrometry Scores. J Endourol. 2011; 25(12):1921-1924.

17. Leibovich BC, Blute M, Cheville JC, et al. Nephron sparing surgery for appropriately selected renal cell carcinoma between 4 and 7 cm results in outcome similar to radical nephrectomy. J Urol. 2004; 171(3):1066-70.

18. Pahernik S, Roos F, Röhrig B, et al. Elective nephron sparing surgery for renal cell carcinoma larger than 4 cm. J Urol. 2008; 179(1):71-4.

19. Veccia A, Antonelli A, Uzzo RG, et al. Predictive Value of Nephrometry Scores in Nephron-sparing Surgery: A Systematic Review and Meta-analysis. Eur Urol Focus. 2020; 6(3):490-504.

10. Kutikov A, Uzzo RG. The R. E. N. A. L. nephrometry score: a comprehensive standardized system for quantitating renal tumor size, location and depth. J Urol 2009; 182:844-853.

20. Bylund JR, Gayheart D, Fleming T, et al. Association of tumor size, location, R. E. N. A. L., PADUA and centrality index score with perioperative outcomes and postoperative renal function. J Urol. 2012; 188:1684-1689.

11. Ficarra V, Novara G, Secco S, et al. Preoperative aspects and dimensions used for an anatomical (PADUA) classification of renal tumours in patients who are candidates for nephron-sparing surgery.

Eur Urol. 2009; 56:786-793.

21. Buffi NM, Saita A, Lughezzani G, et al. Robot-assisted partial nephrectomy for complex (PADUA Score 10) tumors: techniques and results from a multicenter experience at four high-volume centers. Eur Urol. 2020; 77:95-100.

12. Simmons MN, Ching CB, Samplaski MK, et al. Kidney tumor location measurement using the C index method. J Urol. 2010; 183:1708-1713.

22. Sharma AP, Mavuduru RS, Bora GS, et al. Comparison of RENAL, PADUA, and C-index scoring systems in predicting perioperative outcomes after nephron sparing surgery. Indian J Urol. 2018; 34(1):51-55.

13. Spaliviero M, Poon BY, Karlo CA, et al. An Arterial Based Complexity (ABC) scoring system to assess the morbidity profile of partial nephrectomy. Eur Urol. 2016; 69:72-9.

23. Monn MF, Gellhaus PT, Masterson TA, et al. R.E.N.A.L. Nephrometry scoring: how well correlated are urologist, radiologist, and collaborator scores? J Endourol. 2014; 28(8):1006-10.

14. Sempels M, Ben Chehida MA, Meunier P, Waltregny D. Open and Laparoscopic Partial Nephrectomy: Comparison and Validation of Preoperative Scoring Systems, Including PADUA, RENAL, ABC Nephrometric Scores and Perinephric Fat Evaluation with Mayo Adhesive Probability Score. Res Rep Urol. 2021; 19,13: 509-517.

24. Benadiba S, Verin AL, Pignot G, et al. Are urologists and radiologists equally effective in determining the RENAL Nephrometry score? Ann Surg Oncol. 2015; 22(5):1618-24.

15. Yeon JS, Son SJ, Lee YJ, et al. The nephrometry score: Is it effective for predicting perioperative outcome during robot-assisted partial nephrectomy? Korean J Urol. 2014; 55: 254-9.

25. Dahlkamp L, Haeuser L, Winnekendonk G, et al. Interdisciplinary Comparison of PADUA and R.E.N.A.L. Scoring Systems for Prediction of Conversion to Nephrectomy in Patients with Renal Mass Scheduled for Nephron Sparing Surgery. J Urol. 2019; 202(5):890898.

16. Lau WK, Blute ML, Weaver AL, et al. Matched comparison of radical nephrectomy vs nephron sparing surgery in patients with unilateral renal cell carcinoma and a normal contralateral kidney. Mayo Clin Proc. 2000; 75:1236.

26. Dell’Atti L, Scarcella S, Manno S, et al. Approach for Renal Tumors With Low Nephrometry Score Through Unclamped Sutureless Laparoscopic Enucleation Technique: Functional and Oncologic Outcomes. Clin Genitourin Cancer. 2018; 16(6):1251-1256.

CORRESPONDENCE Simone Scarcella Department of Urology, Azienda Ospedaliera “Ospedali Riuniti” 71 Conca Street, 60126, Ancona – Italy E-mail: simoscarc@gmail.com Phone. +39 3924677442

50

Advances in Urological Diagnosis and Imaging - 2021; 4,3

REVIEW

Liquid biopsy in Renal Cell Carcinoma: Current and future applications Alessia Cimadamore 1, Simone Scarcella 2, Erika Palagonia 2, Lucio Dell’Atti 2, Andrea Benedetto Galosi 2, Rodolfo Montironi 1.

1

Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy; 2Department of Urology, Marche Polytechnic University, School of Medicine, United Hospitals, Ancona, Italy.

Renal cell carcinoma (RCC) is the seventh most common cancer and the most lethal urological malignancy with 5‐year overall survival of 74%, that decrease to 8% in patients with evidence of distant metastasis upon initial diagnosis. To date, no serum or urine biomarkers are currently available for early diagnosis, for monitoring recurrence and response to therapy. Liquid biopsy is a non-invasive diagnostic assay that can detect tumor molecular alterations at diagnosis, after surgery and during progression. Liquid biopsy includes testing for circulating tumor nucleic acids (DNA and RNA), circulating tumor cells (CTC) and small vesicles. In this paper, we reviewed the current knowledge on circulating tumor DNA and CTC in RCC, focusing on the newly developed and promising techniques, such as the circulating free-DNA methylation analysis, and their potential applications in the future.

SUMMARY

KEY WORDS: Renal cell carcinoma, kidney tumors, liquid biopsy, circulating tumor cells, circulating DNA, biomarkers.

INTRODUCTION

LIQUID

Renal cell carcinoma (RCC) is the seventh most common cancer and the most lethal urological malignancy with 5‐year overall survival of 74%, that decrease to 8% in patients with evidence of distant metastasis upon initial diagnosis (1).

Around 30% of patients have metastases detected at preoperative screening or surgery, termed synchronous metastases. Up to 50% develop metastases after the removal of the primary tumor (at least 3 months and as late as 30 years after primary surgery), called metachronous metastases. The timing and location of metastases in RCC are difficult to predict, which makes surveillance challenging (2).

At present, radiological assessments of renal masses and metastases are insufficient for qualitative characterization of the tumor. Histopathological evaluation is needed for diagnosis, grading and staging of primary tumors; however, in the setting of metastatic disease, no serum or urine biomarkers are currently available to monitor recurrence and response to therapy. During the last decade, multiple genetic alterations of RCC have been associated with prognosis and response to therapy, even though no specific mutations have been associated with sensitivity or resistance to a specific drug.

Liquid biopsy consists in a liquid sample such as whole blood/plasma/urine used for identifying molecular circulating signatures shared with solid tumors. In other words, it can be considered a surrogate material of tissue/cytological sample and can be used as a non-invasive test that allows multiple serial sampling at any stage of disease (3) (Figure 1).

Liquid biopsy has been proposed as a potential strategy to improved stratification of patients for adjuvant therapy trials (4, 5). The main goal of liquid biopsy in this scenario would be the detection of minimal residual disease following intended curative nephrectomy. In other malignancies such as lung cancer, colon cancer, breast cancer and prostate cancer, the development of circulating tumor DNA (ctDNA) assays is intended to evaluate of the presence of specific genetic alterations able to predict response to targeted therapy. Contrariwise, in the therapeutic scenario of RCC there is no predictive biomarker able to select patient for a specific therapy. As consequence, many of the studies published in the literature have focused on the quantification of ctDNA and CTCs as measures of tumor burden and on its correlation with prognosis and development of metastasis (6, 7).

BIOPSY IN RENAL CELL CARCINOMA

Advances in Urological Diagnosis and Imaging - 2021; 4,3

51

A. Cimadamore, S. Scarcella, E. Palagonia, L. Dell’Atti, A. B. Galosi, R. Montironi.

Figure 1. Graphic description of the process during liquid biopsy. (Image reproduced under Creative Commons license from reference 28)

CTDNA IN RENAL CELL CARCINOMA CtDNA has been detected in the plasma and urine of patients with oncocytomas and early-stage clear cell RCCs (8). This finding is noteworthy since liquid biopsy can be potentially implemented to differentiate small renal masses and to guide decision over invasive surgery versus active surveillance. ctDNA has potential as a surveillance biomarker for patients with localized RCC after nephrectomy. In a study of 30 patients with RCC preparing for nephrectomy, ctDNA NGS was used to interrogate 14 commonly mutated genes.(9) Twenty of the 30 patients had detectable somatic mutations in at least one of the 14 genes assessed.This suggests that even the low tumor burdens seen in localized RCC shed detectable quantities of ctDNA. Another study used quantitative real-time PCR to measure the level of ctDNA in 92 patients with clear-cell RCC across different stages of disease (10). Metastatic RCC (mRCC) patients had a higher level of ctDNA compared to localized RCC (6.04 vs. 5.29, p = 0.017).

The authors also showed that recurrence of RCC was associated with higher levels of ctDNA (p = 0.024).

52

Advances in Urological Diagnosis and Imaging - 2021; 4,3

In the metastatic setting, patients with mRCC are monitored for treatment response with a CT chest/abdomen/pelvis every 3-6 months. Recurrent CT scans are time consuming, costly, and expose cancer patients to high levels of radiation. ctDNA has the potential to act as a surrogate for radiographic disease progression in mRCC. Studies suggest that ctDNA can be routinely monitored at set intervals to monitor for disease recurrence (11, 12). Use of ctDNA could decrease the potential harms associated with screening CT scans, including contrast nephropathy and radiation exposure.

Pal and colleagues detected ctDNA in 78.6% of 200 metastatic patients using the Guardant360 plasma assay (Guardant Health), though with a median of one genomic alteration per sample (13). The same authors detected ctDNA in a further 18/34 (53%) metastatic RCC patients and observed a possible correlation between detection and lesion diameter. In a recent study of 34 patients with mRCC, patients with detectable ctDNA had greater radiographic tumor burden than patients with no detectable ctDNA (14). CtDNA-positive mRCC patients had shorter overall survival and progression-free survival on first-line therapy. Among ctDNA-positive patients,

Liquid biopsy in Renal Cell Carcinoma: Current and future applications

ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables (15).

While these findings suggest that ctDNA has the capability to complement or replace frequent CT scans in patients with mRCC, these results are from a small, retrospective study and warrant further evaluation in larger, prospective cohorts.

Overall, in contrast with other tumors, low levels of ctDNA has been found in RCC. Thirty-three percent of patients had evidence for RCC-derived ctDNA, significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. The probability of detecting ctDNA rises with increasing size of the primary tumor and in patients with growth of a tumor thrombus into the renal vein or inferior vena cava. However, consensus concerning ctDNA levels in RCC has yet to be reached; data are conflicting and often not comparable because of the use of different techniques for DNA extraction, different sequencing methods and limit of detection.

METHYLATION

ANALYSIS OF CIRCULATING FREE DNA IN RENAL CELL CARCINOMA A promising reliable biomarker for early detection of RCC is the analysis of CpG island hypermethylation in circulating free DNA (cfDNA). The first study was published in 2013 by Hauser et al. (16) on a small cohort of patients with RCC. In 30 of 35 investigated patients with RCC, at least one of the eight gene tested was methylated within the serum cfDNA. The Area under the receiver operating characteristic (AUROC) curve showed a high specificity for serum cfDNA methylation (range 85-100%) but low sensitivity in single-gene analysis (range 14-54%). Similar results were obtained in 2016 by Skrypkina et al. testing a different set of genes.(17) In 2018, the first genome-wide cfDNA methylation analysis study was conducted by the newly developed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP–seq) method. With this advanced, highly sensitive and costeffective technique, Shen et al. achieved an AUROC curve of approximately 0.9 for detecting and classifying patients with RCC from patients with other tumor types and healthy controls (18). Recently Nuzzo and colleagues (19) validated the cfMeDIP–seq method in a series of RCC and reported the first application of this method on urine samples. Notably, the authors showed an accurate classification of patients across all stages of renal cell carcinoma (RCC) in plasma (AUROC curve 0.99) and an AUROC curve of 0.858 for correctly classifying urine RCC and control samples in a cohort where two-thirds of patients with RCC had localized disease. This assays proved to be capable of detecting early stage tumors, even at lower sequencing depth and ctDNA abundance (20). Of interest is also the analysis of SHOX2 gene methylation which demonstrated to be strongly correlated with an advanced disease stage and risk of death after initial partial or radical nephrectomy (21), so identifying a group of patients who might benefit from an adjuvant treatment or early initiation of a palliative treatment.

CTC

IN

RENAL

CELL CARCINOMA

Few studies are present in the literature concerning CTC in RCC. In general, detection of CTCs requires specific techniques able to overcome problems related to identification and isolation of tumors cells from blood. CellSearch system, the only platform approved by FDA, showed a very low detection rate in patients with localized and metastatic RCC. However, in samples collected at baseline/presurgery, despite the extremely low CTC counts, the presence of even one single eCTC was associated with a shorter PFS (22).

Presence of CTCs has been associated with more aggressive tumor features and worse outcome (23). A greater number of CTCs was found after open radical nephrectomy than after laparoscopic procedures(24). CTC changes over time and correlation with tumor response have been included as secondary outcomes in ongoing trial (NCT02978118) which also will investigate the immunemarker profile evaluating the expressions of PD-1, PD-L1, CTLA-4, CD27, OX40, or LAG3 on isolated CTCs. Contrarily to Cell Search approach, which detects only cells expressing epithelial markers, marker independent CTC capture approach have been developed (25). Among these new methods, antibody cocktail targeting four RCCCTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80% (26). These newly engineered capture platform outperforms the conventional assay that rely exclusively on epithelial markers and may improve the use of CTCs as potential biomarker (27).

CONCLUSIONS Liquid biopsy is a non-invasive test that allows multiple serial sampling at any stage of disease. ctDNA has potential as prognostic factor, predicting recurrence after nephrectomy, as a surveillance biomarker for patients in follow-up, and as biomarker of response during therapy. Cancer-specific DNA methylation changes could enable highly sensitive and low-cost detection, classification and monitoring of cancer. Improving CTC detection in mRCC by developing alternative CTC detection approaches will help unlock their informative content and offer important hints in treatments planning of this disease.

REFERENCES 1. Gupta K, Miller JD, Li JZ, et al. Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): A literature review. Cancer Treatment Reviews. 2008; 34:193-205.

2. Ljungberg B, Bensalah K, Canfield S, et al. EAU guidelines on renal cell carcinoma: 2014 update. Eur Urol. 2015; 67(5):913-24.

3. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid biopsies into the management of cancer. Nature Reviews Clinical Oncology. 2017; 14:531-48.

4. Cimadamore A, Gasparrini S, Massari F, et al. Emerging Molecular Advances in Urological Diagnosis and Imaging - 2021; 4,3

53

A. Cimadamore, S. Scarcella, E. Palagonia, L. Dell’Atti, A. B. Galosi, R. Montironi.

Technologies in Renal Cell Carcinoma: Liquid Biopsy. Cancers (Basel).

2019; 11(2):196.

5. Cimadamore A, Massari F, Santoni M, et al. Molecular characterization and diagnostic criteria of renal cell carcinoma with emphasis on liquid biopsies. Expert Review of Molecular Diagnostics. 2020; 20(2):141-150.

17. Skrypkina I, Tsyba L, Onyshchenko K, et al. Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer. Dis Markers. 2016; 2016.

18. Shen SY, Singhania R, Fehringer G, et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes.

Nature. 2018; 563(7732):579-83.

6. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6(224).

19. Nuzzo PV, Berchuck JE, Korthauer K, et al. Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med. 2020; 26(7):1041-3.

7. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nature Reviews Genetics. 2019; 20(2):71-88.

20. Cimadamore A, Santoni M, Massari F, et al. Liquid biopsies in renal cell carcinoma with focus on epigenome analysis. Annals of translational medicine. China. 2019; 7:S194.

8. Smith CG, Moser T, Mouliere F, et al. Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors. Genome Med. 2020; 12(1).

9. Al-Qassab U, Lorentz CA, Laganosky D, et al. PNFBA-12 Liquid biopst for renal cell carcinoma. J Urol. 2017; 197(4):e913-4.

10. Wan J, Zhu L, Jiang Z, Cheng K. Monitoring of plasma cell-free DNA in predicting postoperative recurrence of clear cell renal cell carcinoma. Urol Int. 2013; 91(3):273-8.

21. Jung M, Ellinger J, Gevensleben H, et al. Cell-free SHOX2 DNA methylation in blood as a molecular staging parameter for risk stratification in renal cell carcinoma patients: A prospective observational cohort study. Clin Chem. 2019; 65(4):559-68.

22. Cappelletti V, Verzoni E, Ratta R, et al. Analysis of single circulating tumor cells in renal cell carcinoma reveals phenotypic heterogeneity and genomic alterations related to progression. Int J Mol Sci. 2020; 21(4):1475.

11.Yamamoto Y, Uemura M, Fujita M, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma. Cancer Sci. 2019; 110(2):617-28.

23. Bluemke K, Bilkenroth U, Meye A, et al. Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma correlates with prognosis. Cancer Epidemiol Biomarkers Prev. 2009; 18(8):2190-4.

12. Corrò C, Hejhal T, Poyet C, et al. Detecting circulating tumor DNA in renal cancer: An open challenge. Exp Mol Pathol. 2017; 102(2):255-61.

24. Haga N, Onagi A, Koguchi T, et al. Perioperative Detection of Circulating Tumor Cells in Radical or Partial Nephrectomy for Renal Cell Carcinoma. Ann Surg Oncol. 2020; 27(4):1272-1281.

13. Pal SK, Sonpavde G, Agarwal N, et al. Evolution of Circulating Tumor DNA Profile from First-line to Subsequent Therapy in Metastatic Renal Cell Carcinoma. Eur Urol. 2017; 72(4):557-564.

14. Maia MC, Bergerot PG, Dizman N, et al. Association of Circulating Tumor DNA (ctDNA) Detection in Metastatic Renal Cell Carcinoma (mRCC) with Tumor Burden. Kidney Cancer. 2017; 1(1):65-70.

15. Bacon JVW, Annala M, Soleimani M, et al. Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma. Clin Genitourin Cancer. 2020; 18(4):322-331.e2.

16. Hauser S, Zahalka T, Fechner G, Müller SC, Ellinger J. Serum DNA hypermethylation in patients with kidney cancer: Results of a prospective study. Anticancer Res. 2013; 33(10):4651-6.

25. Ye Z, Ding Y, Chen Z, et al. Detecting and phenotyping of aneuploid circulating tumor cells in patients with various malignancies.

Cancer Biol Ther. 2019; 20(4):546-51.

26. Bu J, Nair A, Kubiatowicz LJ, et al. Surface engineering for efficient capture of circulating tumor cells in renal cell carcinoma: From nanoscale analysis to clinical application. Biosens Bioelectron. 2020; 162.

27. Cimadamore A, Aurilio G, Nolé F, et al. Update on Circulating Tumor Cells in Genitourinary Tumors with Focus on Prostate Cancer.

Cells. 2020; 9(6):1495.

28. Remon J, García-Campelo R, de Álava E, et al. Liquid biopsy in oncology: a consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology. Clin Transl Oncol 2020; 22, 823-834.

CORRESPONDENCE Alessia Cimadamore Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals.

Via Conca 71 − 60126 Ancona, Italy.

Phone: +390715964805 e-mail: a.cimadamore@staff.univpm.it

54

Advances in Urological Diagnosis and Imaging - 2021; 4,3

C ASE

REPORT

A Diagnostic Challenging: a Retroperitoneal Mass

Andrea Benedetto Galosi 1, Simone Scarcella 1, Erika Palagonia 1, Daniele Castellani 1, Carlo Giulioni 1, Lucio Dell’Atti 1, Alessia Cimadamore 2.

1 2

Department of Urology, Polytechnic University of Marche, School of Medicine, United Hospitals, Ancona, Italy; Section of Pathological Anatomy, Polytechnic University of the Marche, School of Medicine, United Hospitals, Ancona, Italy.

Completing a Renal Cell Carcinoma (RCC) diagnosis can sometimes be difficult due to the lack of specific tumour markers, validated and ready to use in the daily clinical practice (1, 2). Liquid biopsies are gaining consensus among the scientific urological community representing a potential adjunctive tool to diagnose kidney malignancies within the next future (3, 4). We report a challenging diagnosis of an isolated retroperitoneal mass, resulting at final histo-pathological examination an unusual RCC degenerating from the tubular cells within the cystic wall, exophytic and contralateral to the renal parenchyma.

SUMMARY

KEY WORDS: Renal Cell Carcinoma, retroperitoneal mass.

RADIOLOGICAL PICTURE QUIZ A UROLOGY CONSULT WAS DONE FOR A 65 YEARS-OLD FEMALE PATIENT DUE TO THE INCIDENTAL FINDING OF A 40 MM RIGHT RETROPERITONEAL MASS WHILE PERFORMING AN ABDOMINAL ULTRASONOGRAPHY (US) (FIGURE 1); DURING REGULAR ONCOLOGICAL FOLLOW UP, AFTER THE EXCISION OF A THORACIC DORSUM MELANOMA IN 2019. A SUBSEQUENT ABDOMINAL COMPUTED TOMOGRAPHY (CT) SCAN OF THE ABDOMEN REVEALED A SOLID 33X37 MM RETROPERITONEAL MASS DISLOCATING THE INFERIOR VENA CAVA (IVC), AND ADHERING TO A SIMPLE CYSTIC FORMATION OF 44X46 MM ADJACENT TO THE ANTERIOR RIGHT RENAL POLE (FIGURE 2). THE RADIOLOGICAL DIAGNOSTIC STUDY OF THE LESION WAS COMPLETED WITH AN ABDOMINAL MAGNETIC RESONANCE IMAGING (MRI) (FIGURE 3).

Figure 1.

Figure 2.

Advances in Urological Diagnosis and Imaging - 2021; 4,3

55

A.B. Galosi, S. Scarcella, E. Palagonia, D. Castellani, C. Giulioni, L. Dell’Atti, A. Cimadamore

Figure 3.

ACCORDING TO RADIOLOGICAL IMAGES CHARACTERISTICS WHAT - PHEOCHROMOCYTOMA (PHEO) - CLEAR CELL RENAL CELL CARCINOMA (CCRCC) - MIXED EPITHELIAL STROMAL TUMOURS (MEST) - RETROPERITONEAL MELANOMA RECURRENCE

DISCUSSION A Urology consult was done for a 65 years-old female patient due to the incidental finding of a 40 mm right retroperitoneal mass during an abdominal ultrasonography (US) (Figure 1); following a regular oncological check up owing to the excision of a thoracic dorsum melanoma in 2019. The consulting urologist recommended performing an abdominal computed tomography (CT) scan of the abdomen which revealed a solid hyper-vascularized 33x37 mm retroperitoneal mass dislocating the inferior vena cava (IVC).The lesion was adhering to a simple cystic formation of 44x46 mm juxtaposing the anterior right renal pole (Figure 2). A multidisciplinary meeting was held with a Radiologist and an Endocrinologist that excluded the possibility of a pheochromocytoma (PHEO) diagnosis and advocated for the execution of an abdominal Magnetic Resonance Imaging (MRI) (Figure 3). The MRI findings described a solid nodular formation of 40x35 mm adhering to a simple renal cyst of 45 mm. The lesion was characterized as: vascularized, capsulated, with minimum contents of fat; without a defined anatomical plane within the psoas muscle, but not infiltrating the IVC. A laparotomy surgery was performed and the lesion was entirely excised simultaneously with the simple renal cyst, both were contained into the Gerota’s fascia. No postoperative complication occurred and the patient was discharged after three days following surgery. The definitive anatomopathological report concluded with a clear cell renal cell carcinoma (ccRCC) ISUP grade 1, with vascular infiltration in the peritumoral fat, pathological stage pT3a (Figure 4). The follow up CT scan performed at 6 months after surgery did not show any sign of recurrence.

The peculiarity of this case is the origin of the ccRCC. It is suspected to have degenerated from the tubular cells within the cystic wall, exophytic and contralateral to the renal parenchyma, representing an atypical site for these lesions.

56

Advances in Urological Diagnosis and Imaging - 2021; 4,3

IS THE MOST RELIABLE DIAGNOSIS?

Figure 4.

REFERENCES 1. Campagna R, Pozzi V, Spinelli G, et al. The Utility of Nicotinamide N-Methyltransferase as a Potential Biomarker to Predict the Oncological Outcomes for Urological Cancers: An Update.

Biomolecules. 2021; 11(8):1214.

2. Fantone S, Tossetta G, Graciotti L, et al. Identification of multinucleated cells in human kidney cortex: A way for tissue repairing? J Anat.

2021 Nov 15.

3. Montironi R, Lopez-Beltran A, Cimadamore A, et al. What’s the future in uropathology. Urologia. 2021; 88(4):265-266.

4. Cimadamore A, Gasparrini S, Massari F, et al. Emerging Molecular Technologies in Renal Cell Carcinoma: Liquid Biopsy. Cancers (Basel).

2019; 11(2):196.

CORRESPONDENCE Simone Scarcella, MD Department of Urology, Polytechnic University of Marche, School of Medicine, United Hospitals, Ancona, Italy E-mail: simoscarc@gmail.com Phone. +39 3924677442 Fax: +39 071/5963367

Instructions to Authors AIMS AND SCOPE

Advances in Urological Diagnosis and Imaging is a free open access journal. The Journal has the purpose of promote, spread and favorite the scientific knowledge and research in diagnosis and imaging in Urology, Andrology and Nephrology.

Advances in Urological Diagnosis and Imaging publishes every 4 months original articles, reviews, case reports, position papers, guidelines, editorials, abstracts and congress proceedings.

To publish in Advances in Urological Diagnosis and Imaging is free.

The official language of the journal is English.

For papers with national interest because of local contents, in the “Italian Corner” it’s accepted their publication in Italian. The abstract must be in English. The editing of these papers follows the instructions below described.

All accepted paper will be published after a peer reviewed process.

AUTHORS’

RESPONSIBILITIES

Manuscripts are accepted with the understanding that they have not been published or submitted for publication in any other journal.

Authors must submit the results of clinical and experimental studies conducted according to the Helsinki Declaration on clinical research and to the Ethical Code on animal research set forth by WHO (WHO Chronicle 1985; 39:51).

The Authors must obtain permission to reproduce figures, tables and text from previously published material.

Written permission must be obtained from the original copyright holder (generally the Publisher).

Manuscripts must be written in English language in accordance with the “Uniform Requirements for Manuscripts submitted to biomedical journals” defined by The International Committee of Medical Journal Editors (http://www.ICMJE.org).

Manuscripts in Italian language can be published only after translation (expenses will be charged to the Authors).

Manuscripts should be typed double spaced with wide margins.

They must be subdivided into the following sections: Title page

It must contain: a) title; b) a short (no more than 40 characters) running head title;

c) first, middle and last name of each Author without abbreviations; d) University or Hospital, and Department of each Author; e) last name, address and e-mail of all the Authors; f) corresponding Author; g) phone and/or fax number to facilitate communication; h) acknowledgement of financial support; i) list of abbreviations.

SUMMARY

The Authors must submit a long English summary (300 words, 2000 characters). Subheadings are needed as follows: Objective(s), Material and method(s), Result(s), Conclusion(s). After the Summary, three to ten key words must appear, taken from the standard Index Medicus terminology.

TEXT

For original articles concerning experimental or clinical studies, the following standard scheme must be followed: Summary - Key Words - Introduction - Material and Methods - Results - Discussion - Conclusions - References - Tables - Legends - Figures.

Case Repor t should be divided into: Summary Introduction (optional) - Case report(s) - Conclusions References (Discussion and Supplementary Figures, Tables and References can be submitted for publication in Supplementary Materials).

SIZE

OF MANUSCRIPTS

Literature reviews, Editorials and Original articles concerning experimental or clinical studies should not exceed 3500 words with 3-5 figures or tables, and no more than 30 references.

Case reports, Notes on surgical technique, and Letters to the Editors should not exceed 1000 words (Summary included) with only one table or figure, and no more than three references.

No more than five Authors are permitted.

REFERENCES

References must be sorted in order of quotation and numbered with arabic digits between parentheses. Only the references quoted in the text can be listed. Journal titles must be abbreviated as in the Index Medicus. Only studies published on easily retrieved sources can be quoted. Unpublished studies cannot be quoted, however articles “in press” can be listed with the proper indication of the journal title, year and possibly volume. References must be listed as follows: • Journal articles All Authors if there are six or fewer, otherwise the first three, followed by “et al.”. Complete names for Work Groups or Committees. Complete title in the original language. Title of the journal following Index Medicus

rules. Year of publication; Volume number: First page.

Example: Starzl T, Iwatsuki S, Shaw BW, et al. Left hepatic trisegmentectomy. Surg Gynecol Obstet. 1982; 155:21.

• Books Authors - Complete title in the original language. Edition number (if later than the first). City of publication: Publisher, Year of publication.

Example: Bergel DIA. Cardiovascular dynamics. 2nd ed.

London: Academic Press Inc., 1974.

• Book chapters Authors of the chapters - Complete chapter title. In: Book Editor, complete Book Title, Edition number. City of publication: Publisher, Publication year: first page of chapter in the book.

Example: Sagawa K. The use of central theory and system analysis. In: Bergel DH (Ed), Cardiovascular dynamics. 2nd ed. London: Academic Press Inc., 1964; 115.

TABLES

Tables must be aimed to make comprehension of the written text easier. They must be numbered in Arabic digits and referred to in the text by progressive numbers.

Every table must be accompanied by a brief title. The meaning of any abbreviations must be explained at the bottom of the table itself.

FIGURES Figures are also graphics, algorithms, photographs, drawings.

Figures must be numbered and quoted in the text by number.

The meaning of all symbols, abbreviations or letters must be indicated.

Histology photograph legends must include the enlargement ratio and the staining method. Legends must be collected in one or more separate pages.

Please follow these instructions when preparing files: • Do not include any illustrations as part of your text file.

• Do not prepare any figures in Word as they are not workable.

• Line illustrations must be submitted at 600 DPI. • Halftones and color photos should be submitted at a minimum of 300 DPI.

MANUSCRIPT

REVIEW

Only manuscript written according to the above mentioned rules will be considered.

All submitted manuscripts are evaluated by the Editorial Board and/or by two referees designated by the Editors.

The Authors are informed in a time as short as possible on whether the paper has been accepted, rejected or if a revision is deemed necessary.

The Editors reserve the right to make editorial and liter-

ary corrections with the goal of making the article clearer or more concise, without altering its contents.

Submission of a manuscript implies acceptation of all above rules.

MANUSCRIPT

PRESENTATION

Authors must submit their manuscripts (MAC and WINDOWS Microsoft Word are accepted) to the Assistant Editor (dellatti@hotmail.com).

PROOFS

Authors are responsible for ensuring that all manuscripts are accurately typed before final submission.

Galley proofs will be sent to the Corresponding Author.

Proofs should be returned within seven days from receipt.

IMPORTANT TO KNOW PAPERS ON: MEDICAL AND SURGICAL DEVICES, DIAGNOSTIC INSTRUMENTS, REGISTERED DRUGS, DIET SUPPLEMENTS, NUTRACEUTICALS

S.I.E.U.N. guarantees the Authors the publication of the article for scientific purposes completely free of charge.

Each of the Authors is required to declare at the bottom of their article if they have received funding or grants from Sponsors for publication / study.

Papers that contain references to devices (medical and surgical), diagnostic instruments, registered drugs, diet supplements, nutraceuticals must not be used for commercial purposes without the authorization of Edizioni Scripta Manent.

The Authors are required to declare in the Copyright Assignment Form which possible Sponsors could be interested in a commercial use of the reprints.

Sponsor are requested to buy a minimum amount of 100 reprints at a cost of € 1.500 (1 to 4 pages) or € 2.000 (5 to 8 pages).

Prices for the purchase of number of reprints greater than 100 can be negotiated with Edizioni Scripta Manent.

Edizioni Scripta Manent retains copyright for republishing and the distribution rights for commercial purpose.

TRANSLATION

Translation of manuscripts in Italian language is offered on payment.

Translation and reprints can be requested to Edizioni Scripta Manent by e-mail to info@edizioniscriptamanent.eu

Quello che mancava, ora c’è!

icero Arrigo F. G. C

e Alessandro

Colletti

Uno strumento di lavoro intuitivo e dettagliato su 215 nutraceutici, descritti per singole Schede e valutati secondo i principi della Evidence-Based Medicine.

Di grande utilità per chi si occupa anche di nutrizione, alimentazione e nutraceutica, a supporto dell' attività di counseling per i pazienti.

PREZZO: € 120,00 SPESE DI SPEDIZIONE INCLUSE PER INFO SULLE MODALITÀ DI ACQUISTO SCRIVERE A: INFO@EDIZIONISCRIPTAMANENT.EU

seguici anche qui!! www.salutepertutti.it Il podcast della rIvIsta Ogni settimana parliamo di salute, su come trovare piccoli accorgimenti quotidiani per mantenere il nostro bene più prezioso.

La salute non è solo assenza di malattia, ma anche percezione di benessere, sentirsi bene ed occuparsi di cose utili e belle.

Le voci di Salutepertutti.it sono Alessia Bisini e Ruben Cazzola.