US-Guided Fine-Needle Aspiration Cytology of Para- Aortic Lymph Nods Metastasis in Uterine Cervical Cancer: Accuracy and Impact on Clinical Decision Making

Junping Liu Department of Ultrasound Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital Xin Liu Department of Ultrasound Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital Zhengying Guo Department of Pathology Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital Xiaojuan Lv Department of Gynecologic Oncology Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital Weimin Mao Zhejiang Key Laboratory of the Diagnosis & Treatment Technology on Thoracic Oncology Dong Xu Department of Ultrasound Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital Lijing Wang (  wanglj844@zjcc.org.cn ) Department of Ultrasound Cancer Hospital of the University of Chinese Academy of Sciences Zhejiang Cancer Hospital


Introduction
Cervical cancer is the most common female genital tract malignant tumor and seriously endangers women's health and life. It is generally recognized that PALN metastasis involvement is a unfavorable prognostic indicator in cervical cancer patients, modifying treatment plan and associating with overall survival (1,2). Thus, it is essential to accurately evaluate the PALN metastasis status . However, con rmation of PALN metastases still presents a clinical problem. Computed tomography (CT) and magnetic resonance (MR) imaging identify retroperitoneal metastatic lymph nodes in basis of short-axis diameter (3). The weakness of the size-based criterion is that metastasis of normal size is easily missed (False negative) and enlarged reactive lymph nodes are misdiagnosed as metastases (False positive). Therefore, the investigators studied other diagnostic indicators, concerning contrast material used in the lymph nodes(4) and functional imaging method such as DW-MR (5) imaging and PET/CT (5)(6)(7)(8) . Some researchers demonstrated that DWI-MRI is high sensitive and PET/CT is the most speci c and clinically relevant in detecting para-aortic lymphadenopathy from cervical cancer. However, PET/CT is expensive and unavailable in many districts, especially in developing countries. In addition, PET/CT may easily result in some unnecessary surgery and radiation in view of relatively high false-positive for predicting lymph node metastases(8).
Compared with the above non-invasive imaging methods, surgical biopsy is the gold standard for lymph node diagnosis. Lymphadenectomy has high risk and easily results in blood loss, prolonged hospitalization, postoperative adhesion formation (9). In order to avoid these complications and obtain pathological con rmation, more and more oncologists adopt and approve percutaneous image-guided biopsy technique that is routinely used for differential diagnosis, clinical staging, biological analysis and therapeutic strategy. Although CT-guided biopsy has high spatial resolution and visibility to achieve relatively satisfactory samples, the presence of nonportability, ionizing radiation, and high operating cost and time obviously hinder its clinical application (10,11).
Interestingly, US-FNAC is obtaining a important impact owing to real-time precise visualization and tracking, especially when there are vital structures adjacent to target lesion, including abdominal aorta, inferior vena cava, and gastrointestinal tract (12)(13)(14). However, US-FNAC remains underused for accessing the PALN metastasis status.
In our department, interventional ultrasound has been carried out for nearly twenty years and US-FNAC is accepted as a rst-line diagnostic technique for cervical cancer lymph node metastasis. The objective of this study was to investigate diagnostic accuracy of US-FNAC of enlarged PALNs in cervical cancers, emphasizing in uencing factors and impact on treatment strategies. Materials And Methods 2.1 Patients This study was approved by the Ethics Committee of Zhejiang Cancer Hospital and were in accordance with the ethical standards of the 1964 Helsinki Declaration. The need for informed consent from all patients was waived due to the study's retrospective .We used key words "retroperitoneal, " "para-aortic lymph node, " "biopsy, " and "aspiration" in a interventional ultrasound database between January 1, 2010 and December 30,2018. Figure 1 shows the work ow for the inclusion and exclusion of cases. According to the inclusion criteria, 240 cases were excluded from this study due to different conditions. Consequently, we consecutively enrolled 92 patients with PALNs from cervical cancer who were referred to our department for US-FNAC by gynecological oncologists who required pathological results to guide the treatment of patients.
2.2 Ultrasound examination Before performing US-FNAC, each patient underwent a detailed ultrasound examination to determine the feasibility of FNA. The size, number, location, and characteristics of the affected lymph nodes were assessed using GE-E9 5-16MHz and Esaote MyLab Twice (4-12MHz). The locations of the PLANs to be punctured were divided into four regions, levels -, depending on the anatomical structure(2) (Figure 2). The size of nodules was measured in the central section of the cross-sectional image, taking the diameter perpendicular to the abdominal aorta as the length. The lymph nodes with safe puncture path and the most suspicious morphology were considered as suitable puncture targets. During undergoing US-FNAC, necrotic areas should be avoided as much as possible. In order to avoid damaging vessels in the puncture path, color Doppler was used to con rm the safest puncture path.
2.3 US-FNAC procedures Before puncture, all patients performed coagulation status screening (the international standardized ratio was less than 1.6, prothrombin time < 15 s, partial thromboplastin time < 45 s, platelets > 80,000 per mm 3 .) , and provided informed consent for the US-FNAC procedure. The punctures were carried out by 5 radiologists; all were familiar with the FNAC process. The site of puncture was marked on the skin and the area was cleaned with povidone-iodine solution, and Local anesthesia was administered with 2% lidocaine. The US-FNAC was performed a 20-23 gauge needle(Hakko Co, Japan) attached to an empty 5-mL plastic syringe , using negative suction with at least 1 pass( Figure 3). All operators used free hand technique (without biopsy attachment). The presence of on-site cytology evaluation does not be performed in our department. Patients were required in the hospital and were monitored for vital signs and complications.
Cellular material was then expelled onto a glass slide and the slides were immediately immersed in 95% alcohol. In some cases, cell block was carried out. The specimens were sent to the cytology department. The sample were stained using the haematoxylin and eosin staining method, which is the most commonly used for cytochemical staining, changes the refractive index of the cell's ne structure by color. The nal cytology diagnosis were made by the same cellular pathologists with more than 10 years of experience.
2.4 FNAC results categorization and Final diagnosis. The FNAC results were classi ed into the following groups: unsatisfactory/nondiagnostic material for diagnosis(i, inadequate group), benign or reactive change(ii, benign group), atypical cells present but indeterminate for benign and malignancy(iii, undetermined group), suspicious for malignancy(iv, suspicious group), and positive for malignancy(v, malignant group). An unsatisfactory/nondiagnostic result(i) was de ned as a sample consisting only of blood component or acellular specimen. When mature lymphocytes in morphology or linear colloid-like substance(foreign body granuloma) are obtained from aspirated nodules, benign results(ii) are reported. The undetermined results(iii) was de ned as a sample consisting exclusively of a small amount of atypical cell components, but is not conclusive for benign and malignancy. When cytology diagnosis was reported as suspected malignant or unequivocally malignant, we de ned the diagnosis as suspicious for malignancy(iv) or positive for malignancy(v). The unsatisfactory/nondiagnostic results(i) were de ned as inadequate sample, while the remaining results were adequate/satisfactory specimen. Final diagnoses were determined in cases of (i,ii,iii) by repeat biopsy,surgery,imaging,clinical indicators,and clinical follow-up analyses(15).

Data Statistical
Analyses. The cases of malignant(iv,v) were considered true-positive diagnosis. If the cases of (i,ii,iii) presented shrinkage , disappearance, or stationary condition about a period of 12-24 months without speci c treatment by follow-up imaging, or if subsequent rebiopsy or surgery indicated a benign pathologic result, we de ned cases of (i,ii,iii)as a true-negative result. A false-negative result was de ned as (i,ii,iii) for which nodes were obviously enlarged by follow-up imaging or if the malignant results were con rmed by surgery and rebiopsy or if clinical data highly suggested malignant. We consided false-negative result as incorrect diagnosis. The evaluation index of diagnostic test involved speci city, sensitivity, negative predictive value, positive predictive value and accuracy. These parameters were calculated on the basis of the previous de nition.
Continuous variables were expressed as means±standard deviation, and the analysis was performed using ANOVA; categorical variables were presented on the basis of frequency and percentage, and the analysis was performed using a chi-square test or a Fisher exact test. Univariate analysis (chi-square test or a Fisher exact test) was performed to investigate the relationships among experience,age, body mass index, nodal size, nodal number, lesion location, needle size, cell block, nature and period. Multivariate logistic regression analysis was used to decide variables independently concerning the diagnostic yield of sampling. P < 0.05 for the difference was statistically signi cant. All statistical analysis uses SPSS statistical analysis software(version 21.0 for Windows; SPSS, Chicago, IL, USA).

Results
3.1 Clinical general data. There were 24 cervical cancer patients among these excluded cases( Figure 1). In sixteen of the 24 cases, the enlarged lymph nodes that can be showed in CT imaging cannot be visualized by conventional ultrasound. In ve of the 24 cases in whom size of nodes were small(<10mm), the US-FNAC operations were abandoned. There were no safe access path for puncture to help avoid injury to major vessels in three patients. Of the 92 cases, the age ranged from 25 to 80 years old (means:51.5±10.8y). The average size of nodes ranged from 11mm to 28mm (average:17.1±4.0mm). In the 92 cases, they ranged in the body mass index BMI from 14.6 to 32.0 (means:22.0±3.4). In 79 cases (85.9%) , the multiple nodes were present, and a solitary node was present in thirteen cases(14.1%). 20G and 21G ne needles were used to perform the US-FNAC procedure in 60 cases. The biopsies was performed with 22G and 23G needle using suction in 32 cases.
The anatomical grouping of PLANs are divided into left and right distribution by abdominal aorta. The inferior mesenteric artery is taken as the boundary level line and divided into upper and lower region ( Figure 2). There were 55 cases in the left para-aortic region(level : 27 cases and level : 28 cases). There were 37 cases in the right paraaortic region(level : 29 cases and level : 8 cases)( Table 1). 3.2 Cytological ndings and Final diagnosis. The results of cytological diagnosis by the biopsies were as follows: malignant(n=62;67.4%), suspicious(n=11;12.0%), undetermined(n=5;5.4%),benign(n=10;10.9%), and unsatisfactory(n=4;4.3%). The unsatisfactory materials were provided in 4 cases (4.3%), but 5 cases belonged to undetermined group. In 2 undetermined cases and one inadequate case, a repeat US-FNAC was performed, which provided su cient sample for the diagnosis of squamous cell carcinoma. The remaining 3 undetermined cases and 2 inadequate cases were con rmed as malignant by clinical follow-up and imaging for nal diagnosis.
However, the nal benign diagnosis was established in one inadequate cases due to obvious shrinkage by four months follow-up. Therefore among 9 sample, there were 8 malignant(false-negative) and 1 benign cases(truenegative) in nal diagnosis( Table 2).
Ten cases were reported as benign diagnosis (Figure 4a). Three cases of FNAC benign results were performed by subsequent surgical resections for therapeutic and diagnostic purposes and the de nitive malignant diagnosis was rendered. The nal diagnosis of the remaining 7 benign FNAC results were considered as benign diagnosis by clinical follow-up and imaging. There was no a false-positive result in the study. Therefore nal diagnosis in 10 benign sample consisted of 3 malignant(false-negative) and 7 benign cases(true-negative).
Eighty eight (95.7%) biopsy materials were regarded as satisfactory sample by the more than 10 experienced cytopathologist. Of these 73 FNAC malignant specimens, 62 were considered as an unambiguous malignant diagnosis, including 5 cases of adenocarcinoma (Figure 4b), 39 cases of squamous cell carcinoma (Figure 4c), and 18 cases undifferentiated metastatic carcinoma (Figure 4d), while 11 were reported as a probable metastatic carcinoma diagnosis. According to the de nition(iv,v),the 73 cases were all regarded as malignant cases in nal diagnosis(true-positive).
3.3 Accuracy of US-FNAC. Due to different de nitions of false negative and true negative that previous researchers have adopt for assessing diagnostic accuracy(16), we used the following analysis methods to present our data in this study. When we excluded the four inadequate cases in our analyses and regarded undetermined specimens as  (Table 3). Table 4  Therefore,through US-FNAC technique, the therapeutic methods of 74 patients (80.4%) were subjected to affect.

Discussion
In the current study, our results indicate that FNAC is useful as a technique by sonographic guidance for PLANs in cervical cancer, whose diagnostic accuracy is 90.9%. One of advantages is that the needle tip is displayed in real time throughout the procedure and the needle tip always remains within the lesion during sampling, which may minimize the sample being contaminated with blood and extraneous tissue (movie1). Another advantage of ultrasound guidance is that the manipulator can regulate toward almost any anatomical plane by angling and rotating the transducer to avoid visible blood vessels and important organs. When the needle tip will no longer be seen with US during procedure, we can use gently shaking the puncture needle to track the needle tip position(movie2). However, among 24 excluded cases in our study, 16 US-FNAC operations were abandoned because the enlarged lymph nodes that can be showed in CT imaging cannot be visualized by conventional ultrasound. The possible explanations are small nodules, intestinal gas interference and ake growth of PLANs.
All the patients endured the US-FNAC procedure and there was no apparent evidence of complications. Perforation that is a very worrying complication in the process of abdominal FNAC has not occurred, so antibiotics were not used in patients after biopsy. The nding was also reported by earlier studies (12)(13)(14) . In fact, it is di cult to distinguish bowel from mesentery or omentum after transducer compression and so we made no more effort to follow whether there were bowels in the puncture path. On the whole, the US-FNAC procedure is safe under clinical observation.
Similar to EUS-FNA technique(17) the percutaneous US-FNAC for PLANs is thought to be highly challenging technique that is di cult to master and might make an great effort before the operator achieves an acceptable diagnostic yield. That is, the success rate of US-FNAC is directly associated with the radiologists experience.
Experience refers to the years of engaging in interventional ultrasound. The current research found that of the 11 incorrect diagnoses, 9 cases were less than 3 years of experience. It was generally showed that the practitioner probably require a long learning curve to get the skills that contain obtaining a good view of the target node, selecting a suitable size needle and an appropriate FNAC operating technique. Therefore, we advise that the complicated technique should only be performed by those who have been undergone professional biopsy course training. The following research will focus on describing the learning curve of FNAC as a reference for training.
Our nding indicated that the US-FNAC approach in uenced clinical decision making of 74 patients (80.4%). If patients are found to have PLAN enlargement, para aortic irradiation should be given only when the patient has pathological evidence of PALN metastasis, otherwise spinal cord inhibition and intestinal toxicity injury will be brought. In our study, it is encouraging that US-FNAC results was not false positive, which was also reported by Fisher et al (13). If these patients with cytologically benign PALN can be considered for conservative observation, we should integrate clinical and imaging data (15). Three patients with benign cytological diagnosis were diagnosed as metastatic squamous cell carcinoma by surgical biopsy in our study. One possible interpretation for the nding is that the needle passes may not sample the metastases because of microscopic cancer focus. Another possible interpretation is that sampling error might be occurred and it is possible that metastatic PLANs with surgical excision was not the same aspirated lymph node. When the patients are subject to inadequate specimens, the oncologists must combine clinical data and propose US-FNAC once more. Although the treatment decisions based on PET/CT imaging (15) in some patients, it is recommended to obtain pathological evidence before chemoradiotherapy to reduce unnecessary medical disputes.
The strengths of this study contain the relatively many cervical cancer patients with PLANs enlargement,concerning a high proportion with small PLANs in size. In addition, our study is the rst article that evaluates some factors in uencing diagnostic yield in large samples and highlights independent predictor of diagnostic accuracy by multivariate analysis. What is more, this study investigates the role of the US-FNAC technique in clinical decision making.
The study has the following weaknesses. To begin with, this study is retrospective and has selectivity bias. Next, the diagnosis and treatment of some patients are based on clinical follow up and imaging data. Once more, this is a single-center study and the technical level is different. Ultimately, this study does not make an overall survival analysis, which is the focus of the following research.
In conclusion, US-FNAC is a safe, widely and conveniently available technique. The diagnostic accuracy of this technique is high and the main in uencing factor is the experience of the puncturer. Therefore, it is suggested that the operators should accept formal standardized training and quali cation certi cation.

Consent for publication
Not applicable.

Competing interests
No potential con ict of interest was reported by the authors.

Availability of data and materials
The datasets used and/or analyzed during the current study are not publicly available due privacy rules but are available from the rst author on reasonable request at the following email address liujp85@zjcc.org.cn Note.-Final diagnoses were con rmed by at least two experts, including radio-oncologists, imaging specialists, and pathologists (15). US-FNAC=ultrasound guided ne-needle aspiration cytology PALNs=para-aortic lymph nodes

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