Introduction Growing evidence in the literature supports the accuracy of sentinel lymph node (SLN) biopsy in early-stage cervical cancer. One-step nucleic acid amplification (OSNA) is a rapid assay able to detect cytokeratin 19-mRNA in SLNs, and it can be used for intra-operative detection of low-volume metastases. The aim of this study was to evaluate the rate of low-volume metastasis in SLNs detected by OSNA in patients with early-stage cervical cancer. Secondary aims were to define the sensitivity and the negative predictive value of SLN biopsy assessed with OSNA.
Methods After IRB approval, consecutive patients who underwent surgery for International Federation of Gynecology and Obstetrics stage IA1 with lymph-vascular space involvement to IB1 between November 2017 and July 2019 and had SLN biopsy and pelvic lymphadenectomy were included. SLNs were detected with indocyanine-green cervical injection and sent intra-operatively for OSNA.
Results Eighteen patients underwent SLN assessment with OSNA and systematic pelvic lymphadenectomy in the study period. Four (22.2%) patients had unilateral and 14 (77.8%) had bilateral mapping. OSNA detected micro-metastasis in 6/18 (33.3%) patients. All micro-metastases were detected in patients with bilateral SLN mapping. The sensitivity and negative predictive value of SLN in detecting lymph node metastasis with OSNA calculated per pelvic sidewall were 85.7% and 96.1%, respectively. The false negative rate in mapped sidewalls was 14.3%.
Discussion This is the first series entirely processing SLNs for OSNA in early-stage cervical cancer. OSNA is able to intra-operatively detect low-volume metastases in SLNs. Further studies are necessary to confirm the accuracy of this technique and to assess survival implications of low-volume metastases detected by OSNA.
- cervical cancer
- sentinel lymph node
- surgical oncology
- lymphatic metastasis
- neoplasm micrometastasis
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Sentinel lymph node in early-stage cervical cancer is an accurate method to detect low-volume metastasis
Micro-metastases and isolated tumor cells are usually detected by ultra-staging
One-step nucleic acid amplification is a molecular method able to detect low-volume metastasis in 33.3% of patients
With an estimated 570 000 cases and 311 000 deaths in 2018 worldwide, cervical cancer ranks as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women.1 Growing evidence in the literature supports the accuracy of sentinel lymph node (SLN) biopsy in early-stage cervical cancer, particularly in the case of small volume tumors and bilateral detection.2–5 In particular, pathologic ultra-staging of SLNs potentially allows a sensitivity of 100% for the presence of both micro-metastasis and macro-metastasis in pelvic lymph nodes to be achieved.6 Ultra-staging examination, requiring serial sectioning (0.04–0.25 mm intervals) and the application of immunohistochemistry, appears to be more reliable at detecting micro-metastasis than routine hematoxylin and eosin (H&E), but it cannot be performed intra-operatively as it is too cumbersome, time-consuming, and expensive.7 One-step nucleic acid amplification (OSNA) is a rapid assay, able to detect cytokeratin 19-mRNA in SLN, and it can be used for intra-operative detection of macro-metastasis, micro-metastasis, and isolated tumor cells. It consists of a short homogenization step followed by amplification of cytokeratin 19 mRNA directly from the lysate.8 9 The OSNA method is characterized by a quantitative measurement of the target mRNA in a metastatic lymph node, a brief reaction time for the entire process, a high specificity for the target mRNA, and an absence of genomic DNA amplification. This molecular technique has been largely tested to analyze the SLN in breast cancer, but also in endometrial, thyroid and gastrointestinal cancer,8–12 showing promising results in terms of accuracy. Only one study has tested the role of OSNA in cervical cancer by assessing 130 SLNs from 32 patients.13
The primary aim of this study was to evaluate the rate of low-volume metastases in SLN detected by OSNA assay in patients with early-stage cervical cancer. Secondary aims were to define the sensitivity and the negative predictive value of SLN biopsy assessed with OSNA.
This pilot cohort study was approved by the Institutional Review Board. Consecutive patients who underwent surgery at the Division of Gynecologic Oncology, Fondazione Policlinico Agostino Gemelli IRCCS, for International Federation of Gynecology and Obstetrics (FIGO) 200914 stage IA1 with lymph-vascular space involvement to IB1 cervical carcinoma between November 2017 and July 2019 and had SLN biopsy with systematic pelvic lymphadenectomy, were included. All patients had pre-operative histology confirmation of cervical cancer and underwent a pre-operative abdominal MRI scan, pelvic ultrasound scan, and chest x-ray. Examination under anesthesia was performed at the same time as the surgery. Only women with no evidence of enlarged pelvic and para-aortic lymph nodes (ie, with largest axis <10 mm) at the pre-operative MRI scan were submitted to SLN mapping. Patients with SLN mapping failure, who did not undergo systematic pelvic lymphadenectomy, with special histology sub-types (other than squamous cell carcinoma, adenocarcinoma or adenosquamous carcinoma) or patients with SLN analyzed with H&E or ultra-staging were excluded.
Uterine surgery involved radical hysterectomy in patients who did not wish to retain fertility and cervical conization in patients with tumor diameter <2 cm who desired to preserve fertility.
Sentinel lymph node assessment
The technique and method for SLN mapping was the same in all cases. SLN was detected after superficial and deep cervical injections of indocyanine green (diluted with sterile water at 1.25 mg/mL) at 03:00 and 09:00 hours and sent intra-operatively for OSNA analysis. About 15 min after the cervical injection the retroperitoneal space was opened and the lymph nodes were assessed with a near infra-red camera (Olympus, Tokyo, Japan and Striker, Kalamazoo, Michigan, USA in the case of a laparoscopic/open approach or Da Vinci Xi, Intuitive, Sunnyvale, California, USA in the case of a robotic approach). SLN was defined as the indocyanine green-positive pelvic node closest to the tumor; retroperitoneal spaces were explored in the following order to assess the presence of SLN: eternal iliac, interiliac, obturator, common iliac, parametrial, pre-sacral, and low para-aortic area.15 16 All mapped SLNs were removed and carried in dry containers to the pathology laboratory for OSNA analysis within 30 min.
Systematic bilateral pelvic lymphadenectomy was performed following SLN biopsy in all patients. Non-SLNs were examined with routine H&E staining.
All the SLN samples were defatted, weighed, and processed. SLNs were analyzed with a Gene Amplification Detector (RD- 210) and a new reagent (LS60R) (Sysmex, Chuo-ku, Kobe, Japan) using the OSNA method. The OSNA machine processed the whole lymph node lysate. Nodal sections were homogenized in 4 mL of lysing buffer for 90 s at 25 000 rpm using stainless steel blades or for 60 s at 10 000 rpm using LYNOPREP blade set and centrifuged for 1 min at 10 000 × g. Subsequently, cytokeratin 19 mRNA was amplified by reverse-transcription loop-mediated isothermal amplification in the RD-210.17 18
The OSNA report was considered according to the cut-off values established in previous studies12 13 19 20 (negative <160; isolated tumor cells 160–249; micro-metastasis 250–5000; macro-metastasis >5000 cytokeratin 19 copies/μL). RNA processing by OSNA has been previously described.12
The OSNA method was used when the OSNA machine was available; if it was not available, standard ultra-staging with immunohistochemistry was performed to analyze the SLNs.
The first patients who underwent SLN processing with the OSNA method had immunohistochemistry staining for cytokeratin 19 of the primary tumor to verify the reliability of cytokeratin 19-mRNA detection in SLNs, thus excluding the possibility of false negative cases.
The data are presented as medians and range or counts and percentages, as appropriate. Calculations of sensitivity and negative predictive value were performed per pelvic sidewall in order to delineate the concordance of SLNs assessed by OSNA with histopathology of other pelvic lymph nodes. χ2 or Fisher’s exact tests were used to compare continuous and categorical variables. All statistical tests were performed using SPSS statistical software program Version 20.0 (SPSS Inc, Chicago, Illinois, USA). Statistical tests were two-sided and differences were considered significant at a level of p<0.05.
Eighteen patients underwent SLN assessment with OSNA and systematic pelvic lymphadenectomy in the study period. The first 12/12 (100%) consecutive primary cervical tumors were cytokeratin 19-positive at immunohistochemical staining. Figure 1 demonstrates SLN management of patients with early-stage cervical cancer in the study period. The characteristics of the included patients are shown in Table 1. Four (22.2%) patients had unilateral and 14 (77.8%) had bilateral mapping. Thirty-nine SLNs were analyzed using the OSNA method. The median number of removed SLNs was 2 (range 1–4) per patient.
Overall, eight (44.4%) patients had pelvic lymph node metastasis. These were micro-metastases in seven cases (87.5%) (six detected by OSNA and one by conventional H&E; see Table 2 and online supplementary Table 1) and macro-metastasis in one case (12.5%). In 6/8 women (75.0%) the SLN was the only metastatic lymph node. No patient had para-aortic lymph node metastasis.
The mean intra-operative OSNA measurement time was 20 min. OSNA detected micro-metastasis in 6/18 (33.3%) patients. All micro-metastases were detected in patients with bilateral SLN mapping. No macro-metastasis or isolated tumor cells were detected with the OSNA method. The median number of removed pelvic lymph nodes in SLN-positive patients was 24 (range 14–38). The characteristics of patients with SLN positive for micro-metastasis at OSNA are summarized in Table 2. Four of the six women (66.7%) with positive SLNs for micro-metastasis underwent adjuvant treatment with chemo-radiotherapy, while two (33.3%) underwent no adjuvant treatment.
The false negative rate in mapped sidewalls was 14.3%. Overall, two patients (11.1%) had false negative SLNs. In one patient with unilateral SLN mapping, the metastatic lymph node showed a macro-metastasis in the non-mapped pelvic side. In the other patient, who had bilateral SLN mapping, a micro-metastasis was detected at routine H&E staining in a non-SLN. Both patients who were found to have positive non-SLNs underwent adjuvant chemo-radiotherapy. Online supplementary Table 1 shows the features of the patients with false negative SLNs.
The sensitivity and negative predictive value of SLN in detecting lymph node metastasis with OSNA calculated per sidewall were 85.7% and 96.1%, respectively. The overall accuracy of SLN metastasis detection was 96.9% in patients with bilateral detection.
Table 3 shows a comparison of patients with and without micro-metastasis detected by OSNA according to different clinical and pathological characteristics. None of the analyzed characteristics was significantly different in the two groups.
Table 4 shows a comparison of clinico-pathological characteristics and sensitivity/negative predictive values of SLNs analyzed with OSNA and with ultra-staging; the number of patients with tumor ≥2 cm and type of metastases were significantly different in the two groups. Median follow-up time was 11 months (range 4–21). During this period, no patient experienced recurrence or death.
SLN biopsy in early-stage cervical cancer is gaining interest in the gynecologic oncology community as it provides important and additional information compared with systematic lymphadenectomy. It identifies SLNs in unexpected nodal basins21 and it detects low-volume disease such as micro-metastases or isolated tumor cells that would not otherwise have been detected by routine pathologic nodal processing.22 The standard method to process the SLN is represented by ultra-staging, which requires serial sectioning (0.04–0.25 mm intervals) of lymph nodes and the application of immunohistochemistry; nevertheless, it is a time-consuming and expensive technique, and it cannot be applied intra-operatively.7 The OSNA method has been proposed as an effective tool for intra-operative detection of nodal micro-metastasis and macro-metastasis in different solid tumors.8–12 17 19 In this study we present one of the first reports of the use of OSNA to intra-operatively detect micro-metastasis in early-stage cervical cancer. In our study we demonstrated that all of the first 12 consecutive patients expressed cytokeratin 19 in the primary tumor: this immunohistochemistry test was performed to avoid false negative SLN results, in view of discordant literature reports which described cytokeratin 19 expression in 58.4–100% of the primary tumor sites.23–27
The intra-operative detection rate of micro-metastasis in SLNs with the OSNA assay was 33.3% in the 39 SLNs from 18 women in our series; all of these patients had bilateral SLN mapping. Intra-operative information about metastasis in SLNs is of crucial importance as it could guide physicians to tailor patient’s treatment by extending lymphadenectomy to para-aortic basins and abandoning radical surgery.28 Micro-metastases are described in 4–20% of immunohistochemistry-tested lymph nodes in early-stage cervical cancer,7 27 29 30 and they would be missed at routine frozen section analysis of SLNs. This is the rationale for using an accurate method that is intra-operatively able to detect micro-metastases and isolated tumor cells, such as OSNA. The rate of micro-metastasis in SLNs in our series was 33.3% (no isolated tumor cells were detected); this is higher than the micro-metastasis rate in SLNs described in the literature (4–20%).29–31 However, all these reports detected micro-metastasis with the ultra-staging technique. On the other hand, different authors have reported higher rates of micro-metastasis detection in SLNs from early-stage cervical cancer when SLNs were analyzed using molecular methods rather than immunohistochemistry.23 27 In particular, Van Trappen et al23 used real-time reverse transcription polymerase chain reaction to document absolute copy numbers of cytokeratin 19 in lymph nodes from 32 patients with cervical cancer. Cytokeratin 19 transcription was detected in all primary tumors, and 44% of histologically uninvolved lymph nodes had cytokeratin 19 expression by reverse transcription polymerase chain reaction in 16 of the 32 patients. The authors concluded that approximately 50% of early-stage cervical cancers shed tumor cells to the pelvic lymph nodes and that cytokeratin 19 expression was associated with clinico-pathologic features such as grade and lymph-vascular space involvement. Moreover, Wang et al27 reported that the frequency of micro-metastasis detected by reverse transcription polymerase chain reaction for cytokeratin 19 expression in SLNs from 35 patients with early-stage cervical cancer who had no metastases by routine histologic examination was 42.8%. Additionally, all the SLNs that were confirmed to have metastases by conventional histopathologic techniques showed cytokeratin 19 expression by reverse transcription polymerase chain reaction and/or immunohistochemical analyses. The authors concluded that reverse transcription polymerase chain reaction was more sensitive for detecting micro-metastasis in SLNs than immunohistochemistry in patients with early-stage cervical cancer. Therefore, when compared with molecular techniques, immunohistochemistry may underestimate and possibly miss a quota of micro-metastasis in SLNs in early-stage cervical cancer.29–31 However, this has to be confirmed with larger sample studies. With the limitation of the small number of patients, we have compared SLNs analyzed by OSNA and by ultra-staging. A significant difference in the number of different types of metastases was shown, with a higher number of micro-metastases in the OSNA group and a higher number of macro-metastases in the ultra-staging group. This could be due to the larger tumor size in the ultra-staging group, as tumor size is one of the major determinants for lymph node metastasis.5
Only one study has reported the use of OSNA in cervical cancer SLNs.13 First, the authors confirmed that cytokeratin 19 was one of the optimal markers for the OSNA assay in the detection of lymph node metastases in cervical cancer. Second, they defined 250 cytokeratin 19 copies/μL as the optimal cut-off value to define an OSNA-positive SLN for micro-metastasis; however, no further study has confirmed this cut-off value in cervical cancer, therefore prospective validation trials are needed. Lastly, this study reported detection of micro-metastases in five SLNs from 32 women. Nevertheless, SLNs were processed using step sectioning at 2 mm intervals parallel to the short axis of the node and were examined alternatively with histopathology and OSNA, with a potential tissue allocation bias.
On the other hand, cytokeratin 19 detected by OSNA and reverse transcription polymerase chain reaction is not expressed only by cancer metastasis in lymph nodes. Benign epithelial inclusions such as endosalpingiosis may be the cause of a potential false positivity of these molecular tests. Few studies have reported the presence of benign epithelial inclusions in pelvic and/or para-aortic nodes in 4.7% of patients with gynecologic malignancies,32 14% of female cadavers or patients,33 34 and 3.3% of women treated with radical hysterectomy for cervical carcinoma.35 However, more recently, Lopez-Ruiz et al36 reported a total incidence of benign glandular epithelial inclusions of 0.2% (4/2071 pelvic and para-aortic lymph nodes analyzed), suggesting that epithelial inclusions are much rarer than estimated in the previously existing literature. For this reason, we consider that such a low rate of benign inclusions should not be able to invalidate the OSNA method. Unfortunately, it is not possible to use the same nodal tissue for both OSNA and immunohistochemistry as the tissue used for the molecular assays cannot be used for histology and the formalin-fixed and paraffin-embedded tissue required for permanent sections is not suitable for quantitative mRNA measurements.
The most important aspect of detecting micro-metastasis in SLNs is its clinical implications. To date, very few studies have evaluated the prognostic significance of micro-metastasis in early cervical cancer. Cibula et al29 detected 10.1% rate of micro-metastasis from SLNs of 645 patients with cervical cancer and concluded that the presence of micro-metastasis was associated with a significant reduction in overall survival, which was equivalent to patients with macro-metastasis; 82.5% of patients with micro-metastasis underwent adjuvant treatment. A more recent study37 did not show any impact on disease-free survival in cases of micro-metastasis in SLNs (30.8% of women received adjuvant treatment). However, this study was not designed nor powered to answer the question of the impact of low-volume metastasis on oncologic outcomes. Therefore, the prognostic influence of micro-metastasis in early cervical cancer is still under investigation.
With regard to the accuracy of SLNs assessed by OSNA in detecting nodal metastasis, we had two false negative cases (online supplementary Table 1). In one case a macro-metastasis was missed in the non-mapped pelvic side. As described in other studies,2–4 the accuracy of SLN in detecting lymphatic metastases is significantly increased in bilateral mapping and in tumors <2 cm. In our other case, the micro-metastasis was detected in a non-SLN (surprisingly diagnosed with standard H&E), with a negative OSNA report. It would be difficult in this case to ascribe the missed metastasis to the OSNA or to the SLN mapping; however, we had only one false negative case out of 14 patients with bilateral detection (7.1%), in keeping with SLN failure described in other reports.4 Moreover, this patient would have received adjuvant radiotherapy for pathologic finding on cervical tumor28 (online supplementary Table 1).
The main strength of this study is that, to our knowledge, this is the only study processing the entire SLN completely with OSNA. Additionally, we confirmed the reliability of cytokeratin 19 as a marker which is present in primary cervical tumors.
The major limitation of the present study is represented by the fact that SLNs processed with the OSNA assay were lysate and could not be used for histology and immunohistochemistry. This represents a potential bias, as the histological confirmation of micro-metastasis was not feasible but could have confirmed the micro-metastasis rate. However, a recent literature report showed a negligible frequency of benign epithelial inclusions,36 which represents the possible cause of false positive SLNs. Another limitation is the short median follow-up time; a longer follow-up will give important information about the clinical significance of micro-metastasis detected in SLNs by OSNA.
In this preliminary study we demonstrated that OSNA is a molecular method able to intra-operatively detect low-volume metastases in SLNs of patients with early-stage cervical cancer. However, due to the small sample size, further studies are necessary to confirm the accuracy of this technique, which may be of aid in tailoring patient treatment in the era of personalized medicine. Further research is required to understand the survival implications of low-volume metastases in early-stage cervical cancer.
We would like to acknowledge the surgical team of Gynecologic Oncology Division at Policlinico Gemelli IRCCS, Rome (Italy).
Editor's note This paper will feature in a special issue on sentinel lymph node mapping in 2020.
Twitter @frafanfani, @annafagottimd
Presented at The results of this study were accepted as a poster presentation at the ESGO Conference 2019, Athens, Greece, 2-5 November 2019.
Contributors All the authors approved the final version of the manuscript. NB: data curation, methodology, formal data analysis, writing original draft, conceptualization. LPA: data curation, methodology, formal data analysis. GFZ: data curation, methodology, review and editing, senior pathology author. AS: data curation, methodology. MV: data curation, methodology. FI: data curation, methodology, review and editing. VG: data curation, methodology, review and editing. CC: data curation, methodology. VC: methodology, data curation, review and editing. FF: conceptualization, methodology, review and editing, formal data analysis. AF: methodology, data curation, review and editing. GF: methodology, data curation, review and editing. GS: senior author and coordinator of the study, conceptualization, methodology, review and editing, writing original draft.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Commissioned; externally peer reviewed.
Data availability statement Data are available in a public, open access repository. Data are available upon reasonable request.