- Email: [email protected]
Molecular Diagnosis for Nodal Metastasis in Endoscopically Managed Cervical Cancer: The Accuracy of the APTIMA Test to Detect High-risk Human Papillomavirus Messenger RNA in Sentinel Lymph Nodes
Accepted Manuscript Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to detect high risk HPV-mRNA in sentinel lymph nodes Christhardt Köhler, M.D., Xin Le, M.D., Nasuh Utku Dogan, M.D., Tatiana Pfiffer, M.D., Achim Schneider, M.D., M.P.H, Simone Marnitz, M.D., Julia Bertolini, M.D., Giovanni Favero, M.D. PII:
S1553-4650(16)00137-0
DOI:
10.1016/j.jmig.2016.03.003
Reference:
JMIG 2808
To appear in:
The Journal of Minimally Invasive Gynecology
Received Date: 31 January 2016 Revised Date:
2 March 2016
Accepted Date: 3 March 2016
Please cite this article as: Köhler C, Le X, Dogan NU, Pfiffer T, Schneider A, Marnitz S, Bertolini J, Favero G, Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to detect high risk HPV-mRNA in sentinel lymph nodes, The Journal of Minimally Invasive Gynecology (2016), doi: 10.1016/j.jmig.2016.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 2 3 4 5
For consideration for publication in the Journal of Minimally invasive Gynecology
Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to detect high risk HPV-mRNA in sentinel lymph nodes.
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Christhardt Köhler M.D.1, Xin Le M.D.1, Nasuh Utku Dogan M.D.1,2 *,
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Tatiana Pfiffer M.D.1, Achim Schneider M.D., M.P.H3, Simone Marnitz M.D.4, Julia Bertolini M.D.5, Giovanni Favero M.D.1
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TUBITAK Research Scholar, Ankara, Turkey.
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Institute for Cytology and Dysplasia, Fürstenbergkarree, Berlin, Germany
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Department of Radiooncology, University of Cologne, Germany
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Institute for Pathology, MVZ Hanse Histologicum, Hamburg, Germany
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Department of Advanced Operative and Oncologic Gynecology, Asklepios Hospital Hamburg, Germany
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Department of Obstetrics and Gynecology, Akdeniz University, Antalya, Turkey
Content - Number of pages: 14, Figures: 1, Tables: 2., Word count: 2.300.
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Corresponding author:
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Giovanni Favero M.D., PhD.
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Asklepios Hospital Hamburg
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Department of Advanced GynecologicSurgery and Oncology;
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Eissendorfer Pferdeweg 52, 21075 Hamburg, Germany
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049 40 18 18 864566
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[email protected]
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ACCEPTED MANUSCRIPT Running title: Molecular diagnosis for nodal metastasis in cervical cancer.
Key words: cervical cancer; nodal metastasis; molecular diagnostic; HPV-mRNA; endoscopic sentinel node biopsy.
Abstract
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Background: Nodal metastasis is the most important prognostic parameter in cervical cancer (CC). Detection of HPV-E6/E7-mRNA in lymph nodes
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indicates the presence of vital tumor cells, which may have prognostic
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relevance and may potentially substitute the use of frozen section (FS) and
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ultrastaging (US).
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Objectives: To evaluate the feasibility and accuracy of a commercially
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available test to detect E6/E7-mRNA of 14 sub-types of high-risk HPV´s
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(APTIMA®) in the sentinel lymph nodes of CC patients laparoscopically
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operated.
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Methods: We analyzed prospectively 54 women with HPV-positive CC
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submitted to laparoscopic sentinel node biopsy alone or sentinel node biopsy
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followed by systematic pelvic +/- paraaortic endoscopic lymphadenectomy. All
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sentinel lymph nodes (SLNs) underwent sample collection by cytobrush for
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the APTIMA® assay prior to FS. Results obtained with the HPV-mRNA test
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were compared with the definitive histopathological analysis of the SLNs and
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additional lymph nodes removed.
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Results: A total of 125 SLNs (119 pelvic and 6 paraaortic) were excised with
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a mean number of 2.3 SLNs per patient. Final histopathologic analysis
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confirmed nodal metastases in 10 SLNs from 10 different patients (18%). All
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the histologically confirmed metastatic lymph nodes were also HPV-E6/E7-
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mRNA positive, resulting in a sensitivity of 100%. Four histologically-free
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sentinel nodes were positive for HPV-E6/E7-mRNA, resulting in a specificity
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of 96.4%.
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Conclusion: The HPV-E6/E7-mRNA assay in the SLNs of patients with CC is
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feasible and high accurate. The detection of HPV-mRNA in 4 women with
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negative SLNs might denote a shift from microscopic identification of
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metastasis to molecular level. The prognostic value of this findings awaits
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further verification.
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Introduction In early stage cervical cancer, nodal status is the most important prognos-
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tic factor1. In up to 20% of these patients lymph node metastasis will be found
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at the time of surgery2. Therefore, retroperitoneal lymphadenectomy is cur-
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rently considered an integral part of surgical therapy. Knowledge of the lymph
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node status helps to decide if the patient will benefit from surgery or chemo-
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radiation. However, extensive removal of lymph nodes can lead to relevant
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morbidity. Potential complications associated with the procedure may nega-
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tively impact not only the patient´s quality of life but may also have significant
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adverse oncological consequences3. Thus, the sentinel lymph node (SLN)
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concept has been adapted for women diagnosed with cancer of the uterine
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cervix2. The sentinel node biopsy (SNB) allows to reduce complications and
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has the potential to improve the quality of staging: detection of micrometasta-
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ses and/or isolated tumor cells (ITC) through ultrastaging in the dissected
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sentinel lymph nodes4.
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Up to 15% of initially lymph node-negative early-stage cervical cancer
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patients will develop recurrent disease, which is mainly due to cancer cell
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presence in lymphatic and/or connective tissue undetected by conventional
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histopathologic analysis5. In epithelial malignancies, cytokeratin is used as
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marker to label micrometastases or ITCs. However, for detection of cervical
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cancer cells cytokeratin is proved as non-specific and inferior compared to
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HPV-based assays6. Detection of high-risk HPV-E6/E7-mRNA indicates the
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ACCEPTED MANUSCRIPT presence of viable tumor cells and is an important prognostic factor. To illus-
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trate, Durst et al observed that among pN0 patients the hazard ratio for dis-
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ease-recurrence was 3.8 for HPV-mRNA-positive compared to HPV-mRNA-
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negative ones7. However, the system used in this study was complicated,
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time-consuming and expensive. The availability of a commercial test to detect
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mRNA E6 and E7 of 14 different types of high-risk HPV (APTIMA®) motivated
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us to perform a prospective study to evaluate its accuracy for detection of mi-
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crometastases and/or ITC in patients with HPV-positive early-stage cervical
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cancer.
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Patients and Methods
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Following ethical board review approval (Hamburg ethic committee
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PV5000), we conducted a prospective study of women diagnosed with CC
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(FIGO I-II) submitted to laparoscopic retroperitoneal lymphadenectomy and/or
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sentinel node biopsy between November 2014 and August 2015. The trial
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was carried out in the Asklepios Hospital, Hamburg-Harburg, Germany. HPV
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testing was done at the Institute for Cytology and Dysplasia, Berlin, Germany.
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Inclusion criteria were as follows: (a) histologically and molecularly confirmed
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HPV-associated cervical cancer, (b) age older than 18 and younger than 80
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years, (c) no evidence of extrapelvic disease on initial imaging staging and (d)
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bilateral identification of Sentinel Lymph Nodes. Exclusion criteria were as
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follows: (a) no written informed consent, (b) clinical or surgical contraindica-
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tion for surgery, (c) evidence of peritoneal or distant metastasis detected pre-
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operatively or during surgery, (d) molecularly confirmed HPV-negative cancer.
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All women eligible for the study underwent laparoscopic identification and
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harvesting of SLNs alone or followed by comprehensive endoscopic retroperi-
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toneal lymphadenectomy. The primary objective of this study was to evaluate the feasibility and
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the accuracy of the commercially available HR-HPV-E6/E7-mRNA (APTIMA®
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HPV Assay - Hologic, Bedford, Massachusetts, USA) for the detection of me-
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tastasis in the sentinel lymph nodes of patients with HPV-positive CC. We
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determined the sensitivity and false-negative rates of the method. The defini-
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tive histopathological result of systematic lymphadenectomy was considered
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the criterion standard parameter of comparison. The following information was
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collected: age of the patients at diagnosis, body mass index (BMI), histologi-
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cal type and grade of differentiation, extension of nodal dissection, number of
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removed sentinel lymph nodes and total number of lymph nodes, intraopera-
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tive (IO) and postoperative complications, final pathology and result of HR-
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HPV-E6/E7-mRNA detection.
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The SLN Biopsy Technique:
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At the beginning of surgery, the patients were placed in a lithotomy po-
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sition and subjected to cervical injection of 4 cc of Patent Blue. Once the ad-
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ministration of the labeling substance was finished, the patients underwent
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laparoscopic sentinel node biopsy and retroperitoneal lymphadenectomy.
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The first part of the procedure consisted of careful inspection of the
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abdominal cavity and peritoneal washing, followed by the laparoscopic visual
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identification of the SLN. After transperitoneal locating of the blue spots, the
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retroperitoneum was accessed and the spaces were opened. Each colored
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lymph node was separately excised. In patients with tumors >2cm or if there 5
ACCEPTED MANUSCRIPT was no agreement for sentinel concept, transperitoneal bilateral pelvic and, if
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indicated, paraaortic lymphadenectomy was systematically performed folow-
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ing the technique that has already been described8.
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Samples collection for molecular test:
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Prior to surgery, samples for the HPV-mRNA test from the primary tumor
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were collected using the cytobrush technique and cells were suspended in the
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PreservCyt transport medium. Each harvested sentinel lymph node was cut
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lengthwise and a smear was taken using a cytobrush from the cut plane of
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both halves of the sentinel lymph node. To avoid contamination, a fresh scal-
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pel was used for transection of each individual sentinel lymph node. The cyto-
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brush was immersed in PreservCyt transport medium and sent for HPV-
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mRNA analysis.
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Detection of HPV-E6/E7-mRNA in the primary tumor and in the sentinel
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nodes:
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APTIMA® HPV assay (AHPV) is a qualitative test for detecting mRNA ex-
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pressed from 14 different types of high risk HPV viral E6/E7 oncogenes
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(16/18/31/33/35/39/45/51/52/56/58/59/66 and 68). The procedure was carried
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out according to the manufacturer’s instruction. Briefly, a 1 mL aliquot of each
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of the PreservCyt samples was transferred to 2.9 mL of buffered detergent
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solution. A 400 µL aliquot of this mix was then tested on a semiautomatic Di-
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rect Tube Sampling system (Gen-Probe). Assay results were determined on
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the basis of the signal-to-cutoff ratio (S/CO) for the analysis. Specimens with
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an S/CO value of ≥1.0 were considered positive. The results are obtained
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within approximately 20 minutes.
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Sentinel Node Processing and Histopathological Analysis: Sentinel lymph nodes were either sent to frozen section or fixed in neu-
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tral buffered formaldehyde for approximately 24 hours. Following fixation
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lymph nodes were cut perpendicular to the long axis into slices 0.2 cm thick
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and embedded in a paraffin block. Multiple sections were prepared from each
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block. A set of three 4 µ thick sections was cut every 250 µ and stained by
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hematoxylin-eosin. Detection of tumor cells defined a positive SLN. All non-
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sentinel nodes were processed identically by the pathologists, cut into 3 to 4
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mm sections, and submitted for routine staining (hematoxylin-eosin) and eval-
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uation.
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Statistical Analysis:
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Continuous variables are presented as means and standard deviations.
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Categorical variables are presented as numbers of cases or percentages. The
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false-negative rate was defined as the number of procedures with a negative
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SLN divided by the number of procedures in which the sentinel node was
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identified and a positive lymph node was found. Statistical analyses were per-
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formed using SPSS 20.0 for Windows software. Sensitivity was calculated by
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the ROC curve method.
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Results
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During the period of the study, 55 patients with cervical cancer were
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initially selected to participate in the study. One patient was subsequently
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ACCEPTED MANUSCRIPT identified as not meeting the inclusion criteria and was precluded from the
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trial: this patient had an HPV-negative adenocarcinoma of gastric type. All 54
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women included in the study underwent laparoscopic SNB, followed by endo-
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scopic pelvic +/- PA lymphadenectomy (10 cases). Epidemiological and his-
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tological features of the included patients are summarized in the Table 1. The
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median age of the patients was 42 +13 years (interquartile range = 24 - 72
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years), whereas BMI was, on average, 26 kg/m2 (range, 22 - 48 kg/ m2). His-
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tology revealed squamous-cell carcinoma (SCC) in 44 cases (81%), whereas
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adenocarcinoma was found in the other 10 women (19%). Nine patients
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(16%) had well-differentiated carcinoma, 27 women (50%) presented with
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moderately differentiated tumors, and 18 patients (34%) had poorly differenti-
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ated carcinoma. The distribution of the stages according to the revised 2009
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FIGO staging system for cervical cancer was as follows: Ia2, n =12 (22%);
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Ib1, n = 27 (50%); Ib2, n = 5 (10%); IIa, n = 2 (4%); and IIb, n = 8 (14%).
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All patients underwent laparoscopy only. No casualty related to surgery
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or major IO complication occurred. No excessive blood loss or transfusion
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was observed, and the estimated blood loss (EBL) was approximately 90 cc
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(range, 40 - 250 cc). Among the operated women, there was no intraoperative
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complication and postoperatively 4 (7%) patients developed symptomatic pel-
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vic lymphocele necessitating surgical or radiological drainage.
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The results of the final cytological analysis of the peritoneal washings
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were negative for cancer cells in all cases. The total number of removed
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lymph nodes was 1160 (mean, 22) including the SLNs. In the pelvic (external
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iliac, internal iliac, obturator, common iliac) and the PA regions, 1034 (mean,
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ACCEPTED MANUSCRIPT 19) and, respectively, 126 (mean, 12) lymph nodes were dissected. Sentinel
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nodes were identified in all patients. All women had at least 1 sentinel node in
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each hemipelvis and 6 (11%) patients had also a paraaortic SLN. A total of
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125 SLNs (119 pelvic and 6 PA) were removed, and the mean number of
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SLNs per patient was 2.3 (range, 2 - 4). The results of the final pathology con-
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firmed nodal metastases in 10 SLNs from 10 different patients (18%). The
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sentinel node biopsy had a correlation rate of 100% with the status of the oth-
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er removed nodes (no case of false-negativity). Histologic proved lymph node
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metastases in SLNs were found restricted to the pelvic region in 7 patients
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(70%) and in the PA area in another 3 patients. Histopathologic outcomes are
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demonstrated in the Table 2.
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HPV-E6/E7-mRNA was positive in 11% (14/125) of the removed sen-
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tinel lymph nodes, representing a positivity of 24% (13/54) of the included pa-
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tients. All the histologically confirmed metastatic lymph node (pelvic and
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paraaortic) were also HPV-E6/E7-mRNA positive, resulting in a sensitivity of
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100% (10/10). Additionally, four histologically-free sentinel nodes were posi-
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tive for HPV-E6/E7-mRNA. The results are summarized in the Figure 1.
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Discussion
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Micrometastasis and ITC are rarely identified by conventional histology
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and, consequently, demand advanced and expensive diagnostic methods.
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Regional and systemic cervical cancer metastatic cells display the trans-
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formed genotype induced by HPV in 99% of primary tumors6,9. Thus, detec-
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tion of epithelial cells containing actively transcribed nucleic acids of HPV on-
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cogenes in the lymph nodes may theoretically indicate the presence of meta-
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static cancer cells6. Presently, there are two available types of high-risk HPV nucleic acid
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identification assays: viral DNA detection and, more recently, mRNA-based
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tests. Some previous studies have already investigated the applicability of
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HPV-DNA in predicting nodal metastasis10,11. The authors observed a very
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high sensitivity, but the specificity was very low leading to an unacceptable
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rate of false positive results. Probably, these negative outcomes can be ex-
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plained by the fact that HPV DNA is indeed an inactive form of the virus. The
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detection of HPV DNA in the lymph node may represent DNA from tumor cells
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destroyed by immune competent cells or dead tumor cells, either as circulat-
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ing fragments or internalized by scavenger cells, such as macrophages or
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cellular debris trapped in the lymph node12,13. On the other hand, the HPV-
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E6/E7 gene is essential for the maintenance of the transformed state of the
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cancer cells and, therefore, the identification of HPV-E6/E7-mRNA is directly
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linked only to active forms of the virus. This fact results in a test, detecting
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exclusively viable tumor cells and reducing the rate of false positive results. In
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our study, final pathology of the SLN and the status of HR-HPV-E6/E7-mRNA
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were highly coinciding. All the 10 histologically positive sentinel lymph nodes
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were also HPV-E6/E7-mRNA positive, resulting in a sensitivity of 100%.
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Moreover, among the 111 microscopically negative sentinel nodes, 4 speci-
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men contained HPV mRNA. In the light of current knowledge, one may under-
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stand these results as “false-positive” nodes, resulting in a specificity of
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96.4%. This result is considerably superior compared to those obtained with
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the DNA-HPV technique that varied from 35 to 86%
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10
10,11,12,13
. We hypothesize
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to molecular level identification and it may identify patients with an increased
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risk for recurrence as observed by Dürst et al7. Whereas detection of mi-
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crometastasis may have a prognostic impact, there is a lack of consensus in
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the literature about the predictive value of ITC4. Ultrastaging demands addi-
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tional serial sectioning of the sentinel nodes, and hematoxylin-eosin staining
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combined with immunohistochemistry (IHC) for cytokeratin detection14. As-
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suming that detection of HPV-E6/E7-mRNA in SLN shown to be negative by
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histopathology may be an indirect verification of micrometastasis, the present
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method has a number of technical and financial advantages in comparison to
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conventional IHC.
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Currently, a variety of crucial oncologic information can be obtained
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through the intraoperative histological analysis of the lymph nodes, such as
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that which assists the decision to continue or abandon a radical hysterecto-
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my/trachelectomy, and the extension/level of nodal dissection. Nevertheless,
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the accuracy of frozen sections for detecting nodal metastasis is questionable
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(sensitivity of 81% and false-negative rate of 17%) as observed by Bats et al
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and by Slama et al, respectively15,16. Moreover, it normally demands a com-
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plex organization, highly specialized pathologists, and may considerably pro-
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long the surgical procedure and elevate the costs. With the implementation of
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molecular assays, one can expect a faster (results are obtained within approx-
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imately 20 minutes) and more reliable diagnosis of nodal metastasis. In our
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opinion, another future application of the method is to substitute the use of
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frozen section for nodal examination in the therapy of CC. In this case, the
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use of this technology may have a great impact especially in underserved re-
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gions oft the world were there is a higher incidence of cervical cancer and
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specialized pathologists are rarely available. In conclusion, we have demonstrated that HPV-E6/E7-mRNA assay is a
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feasible method that can correctly predict the status of the lymph nodes in
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patients with initial cervical cancer. Detection of HPV-mRNA must be consid-
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ered as a signal of viable cancer cells in the examined tissue. The commer-
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cially available test can simplify and expand the execution of intraoperative
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analysis of the lymph nodes. Moreover, it has the potential to substitute ul-
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trastaging. The unequivocal clinical utility of the method can significantly im-
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prove the quality of cervical cancer management especially in underserved
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regions. However, our conclusion should be regarded as preliminary since the
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number of examined subjects is limited. Larger validation trials requiring quali-
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ty sentinel node biopsy and retroperitoneal lymphadenectomy are essential to
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properly evaluate the accuracy of the technique.
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Conflict of interest statement: The authors report no conflict of interest.
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11 - Czeglédy J, Iosif C, Forslund O, Willén R, Hansson BG. Detection of human papilloma virus DNA in lymph nodes extirpated at radical surgery for cervical cancer is not predictive of recurrence. J Med Virol. 1998 Mar;54(3):183-5. 12 - Baay MF1, Koudstaal J, Hollema H, Duk JM, Burger MP, Quint WG, Stolz E, Herbrink P. Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer. J Clin Pathol. 1997 Nov;50(11):960-1. 13 - Rose BR, Thompson CH, Jiang XM, Tattersall MH, Elliott PM, Dalrymple C, Cossart YE. Detection of human papillomavirus type 16 E6/E7 transcripts in histologically cancer-free pelvic lymph nodes of patients with cervical carcinoma. Gynecol Oncol. 1994 Feb;52(2):212-7.
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ACCEPTED MANUSCRIPT 14 - Euscher ED, Malpica A, Atkinson EN, Levenback CF, Frumovitz M and Deavers MT. Ultrastaging improves detection of metastases in sentinel lymph nodes of uterine cervix squamous cell carcinoma. The American journal of surgical pathology. 2008; 32:1336-1343.
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15 - Bats AS, Buénerd A, Querleu D, Leblanc E, Daraï E, Morice P, Marret H, Gillaizeau F, Mathevet P, Lécuru F; SENTICOL collaborative group. Diagnostic value of intraoperative examination of sentinel lymph node in early cervical cancer: a prospective, multicenter study. Gynecol Oncol. 2011 Nov;123(2):230-5.
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16 - Slama J, Dundr P, Dusek L, Cibula D. High false negative rate of frozen section examination of sentinel lymph nodes in patients with cervical cancer. Gynecol Oncol. 2013 May;129(2):384-8.
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Legends
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Figure 1 - Association of the HPV-mRNA status with final conventional Histo-
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pathology and HPV mRNA status in sentinel lymph nodes according to topog-
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raphy.
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Table 1 - Epidemiologic and histologic features of the patients with CC in-
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cluded in the study.
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Table 2 - Histopathologic outcomes of the patients with CC included in the
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study
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Median age (years)
42
(24 – 72)
BMI (Kg/m2)
26 (22 – 48)
FIGO-Stage
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Ia2 – 12 (22%) Ib1 – 27 (50%) Ib2 – 5 (10%) IIa – 2 (4%) IIb – 8 (14%) Histology Squamous cell carcinoma
Tumor grading G1
10 (19%) 44 (81%)
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Adenocarcinoma
9 (16%) 27 (50%) 18 (34%)
G2
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Table 1 - Epidemiologic and histologic features of the patients with
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CC included in the study.
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Results Number of dissected nodes
1160 (mean 22)
Number of pelvic nodes
1034 (mean, 19)
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(54 patients) Number of paraaortic nodes
126 ( mean, 12)
(10 patients) Total number of SLNs
125 (mean, 2.3) 119
Paraaortic
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node metastases Number of metastatic SLNs Pelvic
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Paraaortic
18% (10 cases)
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Histologically confirmed lymph
10 7 3
Table 2 - Histopathologic outcomes of the patients with CC
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included in the study
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Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to
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detect high risk HPV-mRNA in sentinel lymph nodes.
Précis:
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The HPV-E6/E7-mRNA assay in the SLNs of patients with cervical
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cancer endoscopically managed is feasible and highly accurate.
S1553-4650(16)00137-0
DOI:
10.1016/j.jmig.2016.03.003
Reference:
JMIG 2808
To appear in:
The Journal of Minimally Invasive Gynecology
Received Date: 31 January 2016 Revised Date:
2 March 2016
Accepted Date: 3 March 2016
Please cite this article as: Köhler C, Le X, Dogan NU, Pfiffer T, Schneider A, Marnitz S, Bertolini J, Favero G, Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to detect high risk HPV-mRNA in sentinel lymph nodes, The Journal of Minimally Invasive Gynecology (2016), doi: 10.1016/j.jmig.2016.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 2 3 4 5
For consideration for publication in the Journal of Minimally invasive Gynecology
Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to detect high risk HPV-mRNA in sentinel lymph nodes.
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Christhardt Köhler M.D.1, Xin Le M.D.1, Nasuh Utku Dogan M.D.1,2 *,
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Tatiana Pfiffer M.D.1, Achim Schneider M.D., M.P.H3, Simone Marnitz M.D.4, Julia Bertolini M.D.5, Giovanni Favero M.D.1
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*
TUBITAK Research Scholar, Ankara, Turkey.
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Institute for Cytology and Dysplasia, Fürstenbergkarree, Berlin, Germany
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Department of Radiooncology, University of Cologne, Germany
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Institute for Pathology, MVZ Hanse Histologicum, Hamburg, Germany
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Department of Advanced Operative and Oncologic Gynecology, Asklepios Hospital Hamburg, Germany
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Department of Obstetrics and Gynecology, Akdeniz University, Antalya, Turkey
Content - Number of pages: 14, Figures: 1, Tables: 2., Word count: 2.300.
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Corresponding author:
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Giovanni Favero M.D., PhD.
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Asklepios Hospital Hamburg
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Department of Advanced GynecologicSurgery and Oncology;
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Eissendorfer Pferdeweg 52, 21075 Hamburg, Germany
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049 40 18 18 864566
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[email protected]
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ACCEPTED MANUSCRIPT Running title: Molecular diagnosis for nodal metastasis in cervical cancer.
Key words: cervical cancer; nodal metastasis; molecular diagnostic; HPV-mRNA; endoscopic sentinel node biopsy.
Abstract
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Background: Nodal metastasis is the most important prognostic parameter in cervical cancer (CC). Detection of HPV-E6/E7-mRNA in lymph nodes
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indicates the presence of vital tumor cells, which may have prognostic
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relevance and may potentially substitute the use of frozen section (FS) and
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ultrastaging (US).
48
Objectives: To evaluate the feasibility and accuracy of a commercially
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available test to detect E6/E7-mRNA of 14 sub-types of high-risk HPV´s
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(APTIMA®) in the sentinel lymph nodes of CC patients laparoscopically
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operated.
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Methods: We analyzed prospectively 54 women with HPV-positive CC
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submitted to laparoscopic sentinel node biopsy alone or sentinel node biopsy
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followed by systematic pelvic +/- paraaortic endoscopic lymphadenectomy. All
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sentinel lymph nodes (SLNs) underwent sample collection by cytobrush for
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the APTIMA® assay prior to FS. Results obtained with the HPV-mRNA test
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were compared with the definitive histopathological analysis of the SLNs and
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additional lymph nodes removed.
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Results: A total of 125 SLNs (119 pelvic and 6 paraaortic) were excised with
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a mean number of 2.3 SLNs per patient. Final histopathologic analysis
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confirmed nodal metastases in 10 SLNs from 10 different patients (18%). All
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the histologically confirmed metastatic lymph nodes were also HPV-E6/E7-
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mRNA positive, resulting in a sensitivity of 100%. Four histologically-free
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sentinel nodes were positive for HPV-E6/E7-mRNA, resulting in a specificity
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of 96.4%.
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Conclusion: The HPV-E6/E7-mRNA assay in the SLNs of patients with CC is
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feasible and high accurate. The detection of HPV-mRNA in 4 women with
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negative SLNs might denote a shift from microscopic identification of
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metastasis to molecular level. The prognostic value of this findings awaits
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further verification.
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Introduction In early stage cervical cancer, nodal status is the most important prognos-
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tic factor1. In up to 20% of these patients lymph node metastasis will be found
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at the time of surgery2. Therefore, retroperitoneal lymphadenectomy is cur-
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rently considered an integral part of surgical therapy. Knowledge of the lymph
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node status helps to decide if the patient will benefit from surgery or chemo-
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radiation. However, extensive removal of lymph nodes can lead to relevant
80
morbidity. Potential complications associated with the procedure may nega-
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tively impact not only the patient´s quality of life but may also have significant
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adverse oncological consequences3. Thus, the sentinel lymph node (SLN)
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concept has been adapted for women diagnosed with cancer of the uterine
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cervix2. The sentinel node biopsy (SNB) allows to reduce complications and
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has the potential to improve the quality of staging: detection of micrometasta-
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ses and/or isolated tumor cells (ITC) through ultrastaging in the dissected
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sentinel lymph nodes4.
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Up to 15% of initially lymph node-negative early-stage cervical cancer
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patients will develop recurrent disease, which is mainly due to cancer cell
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presence in lymphatic and/or connective tissue undetected by conventional
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histopathologic analysis5. In epithelial malignancies, cytokeratin is used as
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marker to label micrometastases or ITCs. However, for detection of cervical
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cancer cells cytokeratin is proved as non-specific and inferior compared to
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HPV-based assays6. Detection of high-risk HPV-E6/E7-mRNA indicates the
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ACCEPTED MANUSCRIPT presence of viable tumor cells and is an important prognostic factor. To illus-
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trate, Durst et al observed that among pN0 patients the hazard ratio for dis-
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ease-recurrence was 3.8 for HPV-mRNA-positive compared to HPV-mRNA-
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negative ones7. However, the system used in this study was complicated,
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time-consuming and expensive. The availability of a commercial test to detect
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mRNA E6 and E7 of 14 different types of high-risk HPV (APTIMA®) motivated
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us to perform a prospective study to evaluate its accuracy for detection of mi-
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crometastases and/or ITC in patients with HPV-positive early-stage cervical
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cancer.
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Patients and Methods
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Following ethical board review approval (Hamburg ethic committee
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PV5000), we conducted a prospective study of women diagnosed with CC
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(FIGO I-II) submitted to laparoscopic retroperitoneal lymphadenectomy and/or
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sentinel node biopsy between November 2014 and August 2015. The trial
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was carried out in the Asklepios Hospital, Hamburg-Harburg, Germany. HPV
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testing was done at the Institute for Cytology and Dysplasia, Berlin, Germany.
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Inclusion criteria were as follows: (a) histologically and molecularly confirmed
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HPV-associated cervical cancer, (b) age older than 18 and younger than 80
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years, (c) no evidence of extrapelvic disease on initial imaging staging and (d)
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bilateral identification of Sentinel Lymph Nodes. Exclusion criteria were as
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follows: (a) no written informed consent, (b) clinical or surgical contraindica-
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tion for surgery, (c) evidence of peritoneal or distant metastasis detected pre-
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operatively or during surgery, (d) molecularly confirmed HPV-negative cancer.
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All women eligible for the study underwent laparoscopic identification and
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harvesting of SLNs alone or followed by comprehensive endoscopic retroperi-
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toneal lymphadenectomy. The primary objective of this study was to evaluate the feasibility and
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the accuracy of the commercially available HR-HPV-E6/E7-mRNA (APTIMA®
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HPV Assay - Hologic, Bedford, Massachusetts, USA) for the detection of me-
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tastasis in the sentinel lymph nodes of patients with HPV-positive CC. We
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determined the sensitivity and false-negative rates of the method. The defini-
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tive histopathological result of systematic lymphadenectomy was considered
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the criterion standard parameter of comparison. The following information was
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collected: age of the patients at diagnosis, body mass index (BMI), histologi-
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cal type and grade of differentiation, extension of nodal dissection, number of
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removed sentinel lymph nodes and total number of lymph nodes, intraopera-
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tive (IO) and postoperative complications, final pathology and result of HR-
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HPV-E6/E7-mRNA detection.
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The SLN Biopsy Technique:
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At the beginning of surgery, the patients were placed in a lithotomy po-
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sition and subjected to cervical injection of 4 cc of Patent Blue. Once the ad-
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ministration of the labeling substance was finished, the patients underwent
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laparoscopic sentinel node biopsy and retroperitoneal lymphadenectomy.
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The first part of the procedure consisted of careful inspection of the
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abdominal cavity and peritoneal washing, followed by the laparoscopic visual
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identification of the SLN. After transperitoneal locating of the blue spots, the
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retroperitoneum was accessed and the spaces were opened. Each colored
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lymph node was separately excised. In patients with tumors >2cm or if there 5
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indicated, paraaortic lymphadenectomy was systematically performed folow-
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ing the technique that has already been described8.
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Samples collection for molecular test:
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Prior to surgery, samples for the HPV-mRNA test from the primary tumor
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were collected using the cytobrush technique and cells were suspended in the
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PreservCyt transport medium. Each harvested sentinel lymph node was cut
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lengthwise and a smear was taken using a cytobrush from the cut plane of
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both halves of the sentinel lymph node. To avoid contamination, a fresh scal-
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pel was used for transection of each individual sentinel lymph node. The cyto-
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brush was immersed in PreservCyt transport medium and sent for HPV-
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mRNA analysis.
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Detection of HPV-E6/E7-mRNA in the primary tumor and in the sentinel
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nodes:
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APTIMA® HPV assay (AHPV) is a qualitative test for detecting mRNA ex-
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pressed from 14 different types of high risk HPV viral E6/E7 oncogenes
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(16/18/31/33/35/39/45/51/52/56/58/59/66 and 68). The procedure was carried
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out according to the manufacturer’s instruction. Briefly, a 1 mL aliquot of each
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of the PreservCyt samples was transferred to 2.9 mL of buffered detergent
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solution. A 400 µL aliquot of this mix was then tested on a semiautomatic Di-
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rect Tube Sampling system (Gen-Probe). Assay results were determined on
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the basis of the signal-to-cutoff ratio (S/CO) for the analysis. Specimens with
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an S/CO value of ≥1.0 were considered positive. The results are obtained
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within approximately 20 minutes.
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Sentinel Node Processing and Histopathological Analysis: Sentinel lymph nodes were either sent to frozen section or fixed in neu-
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tral buffered formaldehyde for approximately 24 hours. Following fixation
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lymph nodes were cut perpendicular to the long axis into slices 0.2 cm thick
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and embedded in a paraffin block. Multiple sections were prepared from each
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block. A set of three 4 µ thick sections was cut every 250 µ and stained by
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hematoxylin-eosin. Detection of tumor cells defined a positive SLN. All non-
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sentinel nodes were processed identically by the pathologists, cut into 3 to 4
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mm sections, and submitted for routine staining (hematoxylin-eosin) and eval-
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uation.
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Statistical Analysis:
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Continuous variables are presented as means and standard deviations.
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Categorical variables are presented as numbers of cases or percentages. The
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false-negative rate was defined as the number of procedures with a negative
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SLN divided by the number of procedures in which the sentinel node was
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identified and a positive lymph node was found. Statistical analyses were per-
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formed using SPSS 20.0 for Windows software. Sensitivity was calculated by
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the ROC curve method.
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Results
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During the period of the study, 55 patients with cervical cancer were
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initially selected to participate in the study. One patient was subsequently
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trial: this patient had an HPV-negative adenocarcinoma of gastric type. All 54
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women included in the study underwent laparoscopic SNB, followed by endo-
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scopic pelvic +/- PA lymphadenectomy (10 cases). Epidemiological and his-
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tological features of the included patients are summarized in the Table 1. The
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median age of the patients was 42 +13 years (interquartile range = 24 - 72
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years), whereas BMI was, on average, 26 kg/m2 (range, 22 - 48 kg/ m2). His-
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tology revealed squamous-cell carcinoma (SCC) in 44 cases (81%), whereas
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adenocarcinoma was found in the other 10 women (19%). Nine patients
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(16%) had well-differentiated carcinoma, 27 women (50%) presented with
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moderately differentiated tumors, and 18 patients (34%) had poorly differenti-
203
ated carcinoma. The distribution of the stages according to the revised 2009
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FIGO staging system for cervical cancer was as follows: Ia2, n =12 (22%);
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Ib1, n = 27 (50%); Ib2, n = 5 (10%); IIa, n = 2 (4%); and IIb, n = 8 (14%).
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All patients underwent laparoscopy only. No casualty related to surgery
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or major IO complication occurred. No excessive blood loss or transfusion
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was observed, and the estimated blood loss (EBL) was approximately 90 cc
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(range, 40 - 250 cc). Among the operated women, there was no intraoperative
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complication and postoperatively 4 (7%) patients developed symptomatic pel-
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vic lymphocele necessitating surgical or radiological drainage.
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The results of the final cytological analysis of the peritoneal washings
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were negative for cancer cells in all cases. The total number of removed
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lymph nodes was 1160 (mean, 22) including the SLNs. In the pelvic (external
215
iliac, internal iliac, obturator, common iliac) and the PA regions, 1034 (mean,
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ACCEPTED MANUSCRIPT 19) and, respectively, 126 (mean, 12) lymph nodes were dissected. Sentinel
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nodes were identified in all patients. All women had at least 1 sentinel node in
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each hemipelvis and 6 (11%) patients had also a paraaortic SLN. A total of
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125 SLNs (119 pelvic and 6 PA) were removed, and the mean number of
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SLNs per patient was 2.3 (range, 2 - 4). The results of the final pathology con-
221
firmed nodal metastases in 10 SLNs from 10 different patients (18%). The
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sentinel node biopsy had a correlation rate of 100% with the status of the oth-
223
er removed nodes (no case of false-negativity). Histologic proved lymph node
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metastases in SLNs were found restricted to the pelvic region in 7 patients
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(70%) and in the PA area in another 3 patients. Histopathologic outcomes are
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demonstrated in the Table 2.
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HPV-E6/E7-mRNA was positive in 11% (14/125) of the removed sen-
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tinel lymph nodes, representing a positivity of 24% (13/54) of the included pa-
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tients. All the histologically confirmed metastatic lymph node (pelvic and
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paraaortic) were also HPV-E6/E7-mRNA positive, resulting in a sensitivity of
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100% (10/10). Additionally, four histologically-free sentinel nodes were posi-
232
tive for HPV-E6/E7-mRNA. The results are summarized in the Figure 1.
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Discussion
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Micrometastasis and ITC are rarely identified by conventional histology
237
and, consequently, demand advanced and expensive diagnostic methods.
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Regional and systemic cervical cancer metastatic cells display the trans-
239
formed genotype induced by HPV in 99% of primary tumors6,9. Thus, detec-
240
tion of epithelial cells containing actively transcribed nucleic acids of HPV on-
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ACCEPTED MANUSCRIPT 241
cogenes in the lymph nodes may theoretically indicate the presence of meta-
242
static cancer cells6. Presently, there are two available types of high-risk HPV nucleic acid
244
identification assays: viral DNA detection and, more recently, mRNA-based
245
tests. Some previous studies have already investigated the applicability of
246
HPV-DNA in predicting nodal metastasis10,11. The authors observed a very
247
high sensitivity, but the specificity was very low leading to an unacceptable
248
rate of false positive results. Probably, these negative outcomes can be ex-
249
plained by the fact that HPV DNA is indeed an inactive form of the virus. The
250
detection of HPV DNA in the lymph node may represent DNA from tumor cells
251
destroyed by immune competent cells or dead tumor cells, either as circulat-
252
ing fragments or internalized by scavenger cells, such as macrophages or
253
cellular debris trapped in the lymph node12,13. On the other hand, the HPV-
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E6/E7 gene is essential for the maintenance of the transformed state of the
255
cancer cells and, therefore, the identification of HPV-E6/E7-mRNA is directly
256
linked only to active forms of the virus. This fact results in a test, detecting
257
exclusively viable tumor cells and reducing the rate of false positive results. In
258
our study, final pathology of the SLN and the status of HR-HPV-E6/E7-mRNA
259
were highly coinciding. All the 10 histologically positive sentinel lymph nodes
260
were also HPV-E6/E7-mRNA positive, resulting in a sensitivity of 100%.
261
Moreover, among the 111 microscopically negative sentinel nodes, 4 speci-
262
men contained HPV mRNA. In the light of current knowledge, one may under-
263
stand these results as “false-positive” nodes, resulting in a specificity of
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96.4%. This result is considerably superior compared to those obtained with
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the DNA-HPV technique that varied from 35 to 86%
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10
10,11,12,13
. We hypothesize
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to molecular level identification and it may identify patients with an increased
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risk for recurrence as observed by Dürst et al7. Whereas detection of mi-
269
crometastasis may have a prognostic impact, there is a lack of consensus in
270
the literature about the predictive value of ITC4. Ultrastaging demands addi-
271
tional serial sectioning of the sentinel nodes, and hematoxylin-eosin staining
272
combined with immunohistochemistry (IHC) for cytokeratin detection14. As-
273
suming that detection of HPV-E6/E7-mRNA in SLN shown to be negative by
274
histopathology may be an indirect verification of micrometastasis, the present
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method has a number of technical and financial advantages in comparison to
276
conventional IHC.
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Currently, a variety of crucial oncologic information can be obtained
278
through the intraoperative histological analysis of the lymph nodes, such as
279
that which assists the decision to continue or abandon a radical hysterecto-
280
my/trachelectomy, and the extension/level of nodal dissection. Nevertheless,
281
the accuracy of frozen sections for detecting nodal metastasis is questionable
282
(sensitivity of 81% and false-negative rate of 17%) as observed by Bats et al
283
and by Slama et al, respectively15,16. Moreover, it normally demands a com-
284
plex organization, highly specialized pathologists, and may considerably pro-
285
long the surgical procedure and elevate the costs. With the implementation of
286
molecular assays, one can expect a faster (results are obtained within approx-
287
imately 20 minutes) and more reliable diagnosis of nodal metastasis. In our
288
opinion, another future application of the method is to substitute the use of
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frozen section for nodal examination in the therapy of CC. In this case, the
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use of this technology may have a great impact especially in underserved re-
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gions oft the world were there is a higher incidence of cervical cancer and
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specialized pathologists are rarely available. In conclusion, we have demonstrated that HPV-E6/E7-mRNA assay is a
294
feasible method that can correctly predict the status of the lymph nodes in
295
patients with initial cervical cancer. Detection of HPV-mRNA must be consid-
296
ered as a signal of viable cancer cells in the examined tissue. The commer-
297
cially available test can simplify and expand the execution of intraoperative
298
analysis of the lymph nodes. Moreover, it has the potential to substitute ul-
299
trastaging. The unequivocal clinical utility of the method can significantly im-
300
prove the quality of cervical cancer management especially in underserved
301
regions. However, our conclusion should be regarded as preliminary since the
302
number of examined subjects is limited. Larger validation trials requiring quali-
303
ty sentinel node biopsy and retroperitoneal lymphadenectomy are essential to
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properly evaluate the accuracy of the technique.
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Conflict of interest statement: The authors report no conflict of interest.
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15 - Bats AS, Buénerd A, Querleu D, Leblanc E, Daraï E, Morice P, Marret H, Gillaizeau F, Mathevet P, Lécuru F; SENTICOL collaborative group. Diagnostic value of intraoperative examination of sentinel lymph node in early cervical cancer: a prospective, multicenter study. Gynecol Oncol. 2011 Nov;123(2):230-5.
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16 - Slama J, Dundr P, Dusek L, Cibula D. High false negative rate of frozen section examination of sentinel lymph nodes in patients with cervical cancer. Gynecol Oncol. 2013 May;129(2):384-8.
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Legends
392 393
Figure 1 - Association of the HPV-mRNA status with final conventional Histo-
394
pathology and HPV mRNA status in sentinel lymph nodes according to topog-
395
raphy.
TE D
EP
396
M AN U
SC
RI PT
368 369 370 371
Table 1 - Epidemiologic and histologic features of the patients with CC in-
398
cluded in the study.
399
AC C
397
400
Table 2 - Histopathologic outcomes of the patients with CC included in the
401
study
14
ACCEPTED MANUSCRIPT
Median age (years)
42
(24 – 72)
BMI (Kg/m2)
26 (22 – 48)
FIGO-Stage
SC
RI PT
Ia2 – 12 (22%) Ib1 – 27 (50%) Ib2 – 5 (10%) IIa – 2 (4%) IIb – 8 (14%) Histology Squamous cell carcinoma
Tumor grading G1
10 (19%) 44 (81%)
M AN U
Adenocarcinoma
9 (16%) 27 (50%) 18 (34%)
G2
TE D
G3
Table 1 - Epidemiologic and histologic features of the patients with
AC C
EP
CC included in the study.
ACCEPTED MANUSCRIPT
Results Number of dissected nodes
1160 (mean 22)
Number of pelvic nodes
1034 (mean, 19)
RI PT
(54 patients) Number of paraaortic nodes
126 ( mean, 12)
(10 patients) Total number of SLNs
125 (mean, 2.3) 119
Paraaortic
SC
Pelvic
6
node metastases Number of metastatic SLNs Pelvic
TE D
Paraaortic
18% (10 cases)
M AN U
Histologically confirmed lymph
10 7 3
Table 2 - Histopathologic outcomes of the patients with CC
AC C
EP
included in the study
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Molecular diagnosis for nodal metastasis in endoscopically managed cervical cancer: the accuracy of APTIMA® test to
RI PT
detect high risk HPV-mRNA in sentinel lymph nodes.
Précis:
SC
The HPV-E6/E7-mRNA assay in the SLNs of patients with cervical
AC C
EP
TE D
M AN U
cancer endoscopically managed is feasible and highly accurate.