Cuadras et al. BMC Cancer (2022) 22:357 https://doi.org/10.1186/s12885-022-09355-0 RESEARCH Open Access Application of One-Step Nucleic Acid Amplification (OSNA) in different cancer entities and usefulness in prostate cancer: a systematic review Mercè Cuadras1, Jacques Planas1* , Ana Celma1, Lucas Regis1, Inés M. de Torres2, M. Eugenia Semidey2, Enrique Trilla1† and Juan Morote1,3† Abstract Background: Lymph node (LN) status is a key prognostic factor in the decision-making process of different cancer entities, including prostate cancer (PCa). Sectioning and haematoxylin and eosin (H&E) staining technique remain the gold standard for the evaluation of LN metastases despite some limitations, especially low sensitivity in detecting an accurate tumour burden within the LN, as well as a subjective and time-consuming result. One-step nucleic acid amplification (OSNA) quantifies mRNA copies of cytokeratin 19 (CK19) in a fast, objective, automated, and reproduc- ible way, raising a general interest to explore its utility for lymphatic metastasis identification in different malignancies. Methods: To present the latest evidence related to the detection of LN metastases in several tumours by using OSNA compared with the conventional H&E method, a systematic review of articles published since March 2021 was con- ducted using PubMed, Cochrane Library, and Web of Science databases. References from primary papers and review articles were checked to obtain further potential studies. Our procedure for evaluating records identified during the literature search followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses criteria. With the aim to design and justify future clinical routine use of OSNA in PCa, novel PCa evidence has been included in this review for the first time. Results: Twenty five studies were included. LN from six different groups of tumours: breast, gastrointestinal, gyneco- logical, lung, head and neck and prostate cancers has been assessed. OSNA was compared with post-operative forma- lin-fixed paraffin-embedded tissue sections with H&E staining as the reference standard. Contingency tables were created, and concordance rate, sensitivity, specificity and predictive values were reported. Seventeen studies analysed the discordant cases using different techniques. Conclusion: OSNA method has a high diagnostic accuracy for the detection of LN metastases in several CK19 expressing tumours. Available evidence might encourage future investigations about its usage in PCa patients to improve LN staging and prognosis. *Correspondence: jplanas@vhebron.net † Enrique Trilla and Juan Morote contributed equally to this work. This work has been realized in the Surgery and Morphological Sciences Doctorate framework of Universitat Autònoma de Barcelona. 1 Urology Department, Vall d’Hebron University Hospital, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cuadras et al. BMC Cancer (2022) 22:357 Page 2 of 10 Keywords: One-step nucleic acid amplification, OSNA, Cytokeratin 19, CK19, Prostate cancer, Lymph node metastases Background low tumour burden in the nodes [11]. High costs, the Prostate cancer (PCa) is the second most incident neo- time required for the analysis, and some limitations to plasm and the fifth cancer specific cause of male mor- standardization have hindered their routine application, tality worldwide [1]. Upon diagnosis, PCa is classified though they remain relevant in clinical research. into major risk categories based on TNM clinical stage, In 2008, an innovative biomolecular technique called biopsy Gleason score, and serum prostate specific anti- One-Step Nucleic Acid Amplification (OSNA) was intro- gen (PSA) levels. High-risk patients associate more bio- duced in Europe to assess LN metastases. OSNA is an chemical recurrence, metastatic progression, and PCa automated system based on reverse transcription loop- related death [2]. mediated isothermal amplification method, able to quan- Pelvic lymph nodes (LN) represent the most common tify copies of cytokeratin 19 (CK19) mRNA. CK19 is a site of metastases in PCa patients considered for surgi- marker expressed by several solid tumours of epithelial cal treatment. According to the series reviewed, the risk origin, but not by healthy lymphatic tissue [12]. OSNA of LN invasion at radical prostatectomy ranges between allows a quick and accurate analysis of the tumour bur- 3 and 24%, and could be even higher in high-risk PCa den of entire LN tissue in an objective, automated, and patients [3]. reproducible way [13–15]. It has been proven useful in Conventional imaging techniques, such as computed different cancer entities, such as breast, colorectal, gas- tomography and magnetic resonance imaging, have low tric, endometrial, cervical, lung, and head and neck sensitivity for the detection of LN metastases [4]. The cancer, achieving a high sensitivity and specificity in introduction of positron emission tomography with dif- the detection of LN involvement, as well as a high con- ferent radiotracers such as 11C-Choline and especially cordance compared to comprehensive histopathological 68Ga-PSMA has increased the sensitivity to detect LN examination, in some cases even comparable to ultra- metastases. The 68Ga-PSMA has demonstrated > 90% staging [16]. specificity with sensitivity rates of 33–99% depending on OSNA was first applied in the intraoperative analysis serum PSA [5]. As ≤5 mm metastases are mostly missed of sentinel lymph node (SLN) in breast cancer, introduc- by these techniques [6], extended pelvic lymph node dis- ing an objective evaluation of the nodal tissue, as well as section (ePLND) remains the most accurate staging pro- reducing the required time and effort by the laboratory cedure despite the fact that up to 20% of patients will personnel. More than 10 years ago, Tsujimoto et al. [15] present some kind of complication after its performance demonstrated the correlation between OSNA and con- [7]. ventional histopathological analysis of the SLN in breast Due to the limited sensitivity of imaging techniques cancer and defined the cut-off values for the distinction in the detection of small metastases, different nomo- between macrometastases, micrometastases, and unaf- grams based on preoperative characteristics have been fected tissue. Since then, more than 200 studies have described in order to define which PCa patient will truly been published and the application range of OSNA was benefit from an ePLND [8, 9]. extended to other cancer entities [17]. Lymphadenectomy extent and histological nodal eval- The available scientific and clinical evidence, together uation have an impact on the staging and consequent with the mentioned characteristics, has introduced prognosis of the disease. The gold-standard procedure OSNA in current national and European clinical guide- consists of a macroscopic identification of the LN, fol- lines as an alternative technique for the determination lowed by its sectioning into 3–4 mm slices, and then of lymphatic involvement in breast cancer through SLN analysis through haematoxylin and eosin (H&E) staining analysis [18]. Moreover, data available from studies in of at least one slice per LN [10]. Main limitations of this colorectal cancer demonstrated that OSNA is a valid approach are metastatic tissue allocation and interob- technique for the detection of lymphatic involvement server bias, as well as being costly and time-consuming. also in this cancer entity [19]. Hence, OSNA is now New methods, such as serial section analysis (slices included in the recommendations for the determination with a thickness of 1–2 mm), immunohistochemistry of biomarkers in colorectal carcinoma [20]. (IHC), and molecular tissue analysis using Reverse Tran- Interestingly, the quantitative outcome of the OSNA scription-Polymerase Chain Reaction (RT-PCR) for PSA assay was identified as useful tool to predict, during sur- have demonstrated a higher sensitivity to identifying gery, non-SLN involvement in breast and gynecological Cuadras et al. BMC Cancer (2022) 22:357 Page 3 of 10 cancer, thus supporting tailoring of surgical procedure Eligible criteria [21]. In breast and colorectal cancer, OSNA was shown We defined study eligibility using the PICO strategy to provide also prognostic information [22]. (patient population, intervention, comparison, and out- Main advantages and disadvantages of OSNA assay comes) [26]. A study was considered relevant to this are summarized in Table 1. review according to the following criteria: 1) Adult Regarding urological tumours, based on previous patients with confirmed cancer, eligible for surgical treat- studies that demonstrated the expression of CK19 in ment and undergoing SLN biopsy (SLNB) or regional PCa tissue, Winter et al. showed that OSNA method lymphadenectomy; 2) patients did not undergo any neo- can detect CK19 mRNA in 100% of primary PCa adjuvant treatment; 3) the main objective was to com- tumours regardless of Gleason score and even more pare OSNA using fresh LN with postoperative standard effectively than CK19 IHC expression, suggesting the formalin-fixed paraffin-embedded (FFPE)-H&E analysis; valid application of this technique in LN evaluation 4) LN were dissected and analysed using both OSNA and [23]. In a very recent study, Engels et al. [24] demon- the standard technique at the same time; 5) the patholog- strated that OSNA can identify nodal metastases at ical examination method was fully described; 6) results an equivalent or, in cases of micrometastases, better were reported per node (minimum 100 nodes); 7) suffi- rate than enhanced histological examination in PCa cient data was available to calculate true-positive, false- patients, confirming its promising use in intraoperative positive, false-negative and true-negative values. We decision-making in personalized LN surgery. limited these criteria to English original studies. Review To set up future clinical use of OSNA in PCa, the aim articles, meta-analysis, conference abstracts, and letters of this review is to analyse the available evidence of this were excluded. technique in different tumours and propose short-term course of actions to transfer the validated concepts and Study selection successes from the other malignancies to PCa. The flow diagram of study selection process was outlined in Fig. 1. A total of 244 potentially relevant studies were identified using the searching terms described. Eighty- Methods nine duplicated studies were initially excluded. After Search strategy screening titles and abstracts, 102 papers were removed. To retrieve all relevant papers published before the From the remaining 52 studies, 28 were excluded after end of March 2021, three databases including PubMed, full text review because the comparison was made with Cochrane Library, and Web of Science were searched intraoperative frozen section or touch imprint cytology by two independent reviewers combining the following as a reference method, less than 100 nodes were included, Medical Subject Headings: one-step nucleic acid ampli- analysis was performed per patient, or insufficient data fication, OSNA, lymph nodes, lymph node metastases, was available to form 2 × 2 tables. cytokeratin 19, CK19. References from primary papers Finally, 25 studies met all the requirements to be con- and review articles were checked to obtain further sidered in the systematic review. potential studies. Our procedure for evaluating records identified during the literature search followed the Quality assessment Preferred Reporting Items for Systematic Reviews and Quality Assessment of Diagnostic Accuracy Studies Meta-analyses (PRISMA) criteria [25]. Disagreements 2 (QUADAS-2) was used as an evidence-based qual- were resolved through discussion. ity assessment tool [27]. QUADAS-2 comprised four domains: patient selection, index test, reference standard, Table 1 Advantages and disadvantages of OSNA Advantages Disadvantages Fast, objective, automated, and reproducible technique Not valid for non-CK19 expressing tumours Intraoperative analysis Trained pathologist needed (thorough dissection) Analysis of the whole LN Potential contamination of the sample Quantitative analysis: Not applicable in case of coexisting neoplasms with the same LN drainage • Cut-off points for macro and micrometastases • TTL: potential predictive and prognostic factor Ability to a more accurate identification of micrometastases No tissue left to re-analysis (except RNA-based molecular tests) Cuadras et al. BMC Cancer (2022) 22:357 Page 4 of 10 Fig. 1 PRISMA flow diagram and flow and timing. The risk of bias of each study was [49] and thyroid cancers [50]— and 6) PCa [24]. All evaluated by two independent reviewers as low “+”, high studies were prospectively designed. OSNA was con- “-” or unclear “?” risk. sidered as index test and a threshold of 250 copies of The QUADAS-2 results summarized in Table 2 suggest CK19 mRNA per μL was fixed to differentiate between a low risk of bias and a moderate to high overall quality of negative (< 250 copies/μL) and positive (≥250 copies/ all 25 included studies. μL) results. OSNA was compared with post-operative FFPE tissue sections with H&E staining as the refer- Results ence standard. Eleven studies also included also CK19 The 25 eligible studies have been published between IHC analysis in addition to H&E staining and OSNA. January 2007 and March 2021. Our review included A LN was cut into at least two parts (depending on LN SLN and non-SLN from six different groups of size) and divided between OSNA assay and pathology. tumours: 1) breast [15, 28–34], 2) gastrointestinal — Contingency tables were created, and concordance rate colorectal [35–38] and gastric cancers [39–41]—, 3) was reported. Seventeen studies analysed the discord- gynecological —cervical [42] and endometrial can- ant cases (OSNA + / H&E -; OSNA - / H&E+) using cers [43–45]—, 4) lung [46–48], 5) head and neck — different techniques. head and neck squamous cell carcinomas (HNSCC) Detailed characteristics are shown in Table 3. Cuadras et al. BMC Cancer (2022) 22:357 Page 5 of 10 Table 2 Risk of bias of included studies a) Assessment of risk of bias. Summary of risk of bias for each study; +: low risk of bias; −: high risk of bias;?: unclear risk of bias b) Risk of bias graph about each risk of bias item presented as percentages across all included studies Cuadras et al. BMC Cancer (2022) 22:357 Page 6 of 10 Table 3 Characteristics of included studies Group Author Year Country Tumour type No. Patients No. Nodes Reference method CK19 IHC Analysis of discordant cases 1) Tsujimoto et al. [15] 2007 Japan Breast 101 325 H&E and IHC yes yes Feldman et al. [28] 2011 USA Breast 496 1044 H&E and IHC yes yes Le Frère-Belda et al. [29] 2012 France Breast 233 503 H&E and IHC yes yes Vegué et al. [30] 2012 Spain Breast 57 567 H&E yes no Wang et al. [31] 2012 China Breast 552 1188 H&E no yes Pathmanathan et al. 2014 Australia Breast 98 170 H&E and IHC no no [32] Banerjee et al. [33] 2014 UK Breast 170 268 H&E no no Li et al. [34] 2015 China Breast 115 311 H&E no yes 2) Croner et al. [35] 2010 Germany Colorectal 184 184 H&E and IHC yes yes Yamamoto et al. [36] 2011 Japan Colorectal 85 385 H&E no yes Guller et al. [37] 2012 Switzerland Colorectal 22 313 H&E and IHC yes yes Yamamoto et al. [38] 2016 Japan Colorectal 204 1925 H&E no no Yaguchi et al. [39] 2011 Japan Gastric 32 162 H&E no yes Kumagai et al. [40] 2014 Japan Gastric 61 394 H&E no yes Shimada et al. [41] 2019 Japan Gastric 43 439 H&E no yes 3) Okamoto et al. [42](29) 2013 Japan Cervical 32 130 H&E no yes Nagai et al. [43] 2015 Japan Endometrial 35 137 H&E no yes Fanfani et al. [44] 2018 Italy Endometrial 40 110 H&E and IHC yes no Kost’un et al. [45](30) 2019 Czech Republic Endometrial 58 135 H&E and IHC yes no 4) Inoue et al. [46] 2012 Japan Lung 49 165 H&E and IHC no no Nakagawa et al. [47] 2016 Japan Lung 111 410 H&E no yes Escalante-Pérez et al. 2019 Spain Lung 160 705 H&E and IHC yes no [48] 5) Goda et al. [49] 2012 Japan HNSCC 65 312 H&E no yes Sofía del Carmen et al. 2016 Spain Thyroid 37 267 H&E and IHC yes yes [50] 6) Engels et al. [24] 2021 Germany Prostate 64 534 H&E and IHC yes yes CK19 Cytokeratin 19, FN False negative, FP False positive, HNSCC Head and neck squamous cell carcinoma, H&E Hematoxylin and eosin, IHC Immunohistochemistry, No Number of, TN True negative, TP True positive Sensitivity, specificity, positive predictive value (PPV), general interest to explore its utility for lymphatic metas- negative predictive value (NPV), and concordance are tases identification in different tumours. listed in Table 4. Discordant cases are included in the OSNA gives a quantitative result of CK19 mRNA cop- reported results. ies, which is present in several simple epithelia but is not expressed in healthy lymphatic tissue [12]. CK19 was ini- Discussion tially proposed as a marker for the detection of LN metas- LN status is a key prognostic factor in the decision- tases in breast cancer, where it is found in up to 98% of making process of cancer management. For a long time, cases [51]. In 2007, Tsujimoto et al. [15] determined 250 sectioning and H&E staining technique has been the copies/μl as the optimum cut-off point to define a posi- gold standard for the evaluation of LN metastases. Even tive axillary LN in breast cancer population. Nonetheless, though it remains an adequate tool, some limitations it is known that the number of positive LN and the size have been described, especially low sensitivity in detect- of metastases are significant prognostic factors in most ing the accurate tumour burden, mainly as a consequence tumours. Therefore, it was also established a second cut-off of sampling bias [10], as well as a subjective and time- point of 5000 copies/μl to distinguish between micro and consuming result. To overcome these limitations, OSNA macrometastases [15]. Subsequent studies have confirmed assay has been developed as a fast, objective, automated, these values and all the results reflected in this review are and reproducible way to examine the whole LN, raising a based on them. Cuadras et al. BMC Cancer (2022) 22:357 Page 7 of 10 Table 4 OSNA accuracy compared with histopathological examination in included tumours Group Author H&E positive H&E negative Sensitivity Specificity Concordance OSNA negative OSNA positive OSNA negative OSNA positive 1) Tsujimoto et al. [15] 0.6 (2) 13.2 (43) 85 (276) 1.2 (4) 95.6 98.6 98.2 (319/325) Feldman et al. [28] 3 (31) 10.2 (107) 83.1 (868) 3.6 (38) 77.5 95.8 93.4 (975/1044) Le Frère-Belda et al. [29] 2.4 (12) 10.1 (51) 82.1 (413) 5.4 (27) 80.9 93.9 92.2 (464/503) Vegué et al. [30] 0 (0) 1.1 (6) 92.1 (522) 6.9 (39) 100 93 93.1 (528/567) Wang et al. [31] 2.6 (31) 13.4 (159) 78 (927) 6 (71) 83.7 92.9 91.4 (1086/1188) Pathmanathan et al. [32] 1.8 (3) 14.7 (25) 80.6 (137) 3 (5) 89.3 96.5 95.3 (162/170) Banerjee et al. [33] 0.7 (2) 14.6 (39) 81 (217) 3.7 (10) 95.1 95.6 95.5 (256/268) Li et al. [34] 2 (6) 9.6 (30) 85.5 (266) 2.9 (9) 83.3 96.7 95.2 (296/311) 2) Croner et al. [35] 1.6 (3) 20.1 (37) 75.5 (139) 2.7 (5) 92.5 96.5 95.7 (176/184) Yamamoto et al. [36] 1 (4) 20.5 (79) 76.6 (295) 1.8 (7) 95.2 97.7 97.1 (374/385) Guller et al. [37] 0.6 (2) 16.3 (51) 79.6 (249) 3.5 (11) 96.2 95.7 95.8 (300/313) Yamamoto et al. [38] 1 (20) 6.5 (125) 89.2 (1717) 3.3 (63) 86.2 96.5 95.7 (1842/1925) Yaguchi et al. [39] 3.1 (5) 24.7 (40) 69.8 (113) 2.5 (4) 88.9 96.6 94.4 (153/162) Kumagai et al. [40] 2.3 (9) 11.4 (45) 82.7 (326) 3.6 (14) 83.3 95.9 94.2 (371/394) Shimada et al. [41] 1.8 (8) 3.2 (14) 93.9 (412) 1.1 (5) 63.6 98.8 97 (426/439) 3) Okamoto et al. [42] 2.3 (3) 2.3 (3) 93.8 (122) 1.5 (2) 50 98.4 96.2 (125/130) Nagai et al. [43] 2.2(3) 10.2 (14) 86.9 (119) 0.7 (1) 82.4 99.2 97.1 (133/137) Fanfani et al. [44] 0.9 (1) 7.2 (8) 86.4 (95) 5.4 (6) 88.9 94.1 93.6 (103/110) Kost’un et al. [45] 0.7 (1) 7.4 (10) 78.5 (106) 13.3 (18) 90.9 85.5 85.9 (116/135) 4) Inoue et al. [46] 0.6 (1) 11.5 (19) 87.3 (144) 0.6 (1) 95 99.3 98.8 (163/165) Nakagawa et al. [47] 2.9 (12) 11.5 (47) 81.2 (333) 4.4 (18) 79.7 94.5 92.7 (380/410) Escalante-Pérez et al. [48] 0.1 (1) 4.8 (34) 91.3 (644) 3.7 (26) 97.1 96.1 96.2 (678/705) 5) Goda et al. [49] 2.6 (8) 17 (53) 77.2 (241) 3.2 (10) 86.9 96 94.2 (294/312) Sofía del Carmen et al. 5.2 (14) 28.1 (75) 60 (160) 6.7 (18) 84.2 89.9 88 (235/267) [50] 6) Engels et al. [24] 2.2 (12) 14.2 (76) 80.5 (452) 3.4 (18) 91.4 100 98.8 (528/534) Figures are expressed as percentages and (number of cases) in parentheses H&E Hematoxylin and eosin, No Number of, OSNA One-step nucleic acid amplification In 2013 V. Peg et al. [21] defined the concept of total a systematic review of the available literature, to date only tumour load (TTL) as the total CK19 mRNA copies of two studies related to PCa have been found [23, 24]. all positive SLNs. TTL serves as a predictive and prog- All the articles included in this review compare OSNA nosis value, providing more accurate staging than path- assay with postoperative H&E staining in the same LN. ological findings. Accordingly, different OSNA studies There is a general concordance between OSNA and in breast SLN have set cut-off values in order to predict standard H&E of over 85%. No full information about the axillary LN status; some of which (10,000–15,000 discordant cases is available, but we have found not only copies) are already included in clinical guidelines [52, different explanations for them but also heterogeneity in 53]. In 2017, Rakislova et al. [54] explored its utility its analysis. Main justifications for the discordant cases to predict recurrences in colorectal carcinoma, and are low or no tumour CK19 expression, tumour alloca- a recent study confirmed that a TTL ≥ 6000 copies/μl tion bias (TAB) and contamination by other epithelial was associated with worse disease free survival in those cells [46]. patients [55]. As CK19 is the single molecular marker used in The analysis of SLN in breast cancer patients is still its OSNA assay, low tumour CK19 expression may result main clinical application, but over the years OSNA has in a false-negative OSNA case. Different CK19 expres- raised interest in the pathology community for a more sion levels have been described for other malignancies accurate LN staging in other cancer entities. In the last such as colorectal (94.1%) [36], gastric (98.6%) [39], decade, several reports comparing OSNA with histo- gynecological (98%) [43], lung (96%) [48], HNSCC pathological examination have been published, but after (91.1%) [49] or PCa (100%) [23]. Moreover, certain Cuadras et al. BMC Cancer (2022) 22:357 Page 8 of 10 tumour subtypes are more likely to the lack of CK19 dysplastic and benign hyperplastic tissues, although com- expression, as observed in metaplastic and lobular plete data concerning CK19-RNA levels is still missing breast carcinomas [56]. Interestingly, Goda et al. [49] [57]. In 2018, Winter et al. [23] published the first OSNA performed a CK19 IHC in primary HNSCC as a first assay in PCa. A total of 20 primary PCa tumours from step when analyzing discordant results, detecting no intermediate-high risk PCa patients (Gleason ≥7) were expression of CK19 in 75% of those cases. Aiming to analysed. A central slice was analysed by OSNA, while reduce false-negative cases in breast, thyroid and lung surrounding slices were sent for both conventional H&E carcinomas, Vegué et al. [30], del Carmen et al. [50], staining and CK19 IHC. PCa was confirmed by H&E in and Escalante Pérez et al. [48] verified the presence of all 20 samples; OSNA was able to detect CK19 mRNA CK19 in primary tumour by IHC before LN analysis. in 100% of cases, ranging from 320 to 250.000copies/μl For a more accurate interpretation of the results, we while IHC did not detect CK19 in one specimen. Given encourage future researchers to include CK19 expres- the small sample size and the high tumour burden of the sion in primary tumours as patient inclusion criteria. selected patients, we cannot fully extrapolate these find- When compared to histopathological examination, ings for all PCa patients. Recently, Engels et al. [24] pub- OSNA offers the advantage to obtain objective and quanti- lished an assay that verifies the reliability of OSNA for tative data about tumour load of the whole LN in a fast and the first time in PCa. A total of 574 SLNs from 64 PCa effortless way, avoiding interobserver variability. However, patients undergoing prostatectomy and sentinel lym- to properly compare both techniques on the same node, phadenectomy were included. SLNs were assessed by it is mandatory to split it, leading to a possible misdetec- conventional H&E staining and OSNA assay. The com- tion of metastasis by one of the methods, which is called parison between both techniques showed a sensitivity, TAB. Trying to justify the discrepancies by the need for specificity and concordance rates of 84.2, 96.1 and 94.4%, sectioning, 17 studies have reported different strategies: respectively, concluding that OSNA assay provides an second-round OSNA analysis, CK19 IHC, exhaustion of accurate diagnosis that might improve LN staging in PCa. the paraffin-embedded SLN slices, CK19 Western blot, or To date, the ePLND remains the most accurate staging CK19 qRT-PCR. Most of the metastases from the discrep- procedure [58], but individual assessment of all dissected ancies were confirmed thanks to these strategies. LNs is laborious and time consuming. Using OSNA, LNs In contrast, a potential disadvantage of examining can be pooled together and analysed in a few samples the whole LN with OSNA is that there is no tissue left as already done in colorectal cancer [54]. In fact, Engels for subsequent histopathological examination follow- et al. [24] suggested that such approach could be feasible ing complete homogenization. Nonetheless, RNA-based to analyse PCa LNs as a faster and economic alternative. molecular tests are possible using OSNA lysate, thus Based on available evidence, current European PCa allowing any follow-up molecular testing. Moreover, Guidelines [58] state a weak recommendation of offering fresh lymphatic tissue requires a thorough dissection to adjuvant therapy to pN1 PCa patients with ≥2 positive avoid missing nodes, which must be completely sepa- LN after radical prostatectomy with ePLND, especially rated from fat tissue by a trained pathologist. It is also in cases with higher pathologic grade, as they have an important to note that in the case of a coexisting neo- increased risk of biochemical recurrence. To that point, plasm, OSNA cannot define which primary tumour the post-operative OSNA analysis and the possibility to metastases come from and is unable to distinguish LN define a TTL value might be helpful in the identification tumour cells from other benign epithelial inclusions. of this subgroup of patients suitable for adjuvant therapy. Therefore, it is imperative to exclude other cancers with Moreover, intraoperative use of OSNA may be a great the same lymphatic drainage and avoid contaminations. opportunity to set up sentinel-guided LN dissection in Despite these concerns, the results reported show a PCa. Future investigations may bring light to the clinical high specificity, concordance rate, and NPV of OSNA impact of OSNA in PCa as well as to its potential predic- assay when compared with the H&E method. Specifically, tive and prognostic roles. in breast cancer patients, that high NPV provides enough evidence to become the gold standard for SLN evaluation. Conclusion As slicing is required for the analysis per node, a concord- OSNA is a suitable tool to standardize LN evaluation in ance of 100% cannot be achieved due to Tab. A high con- most CK19 expressing tumours due to the possibility to cordance rate of over 85% suggests that OSNA could be analyse the whole LN in a fast, objective, automated, and an alternative technique to histopathological examination reproducible way. According to limited data available, in terms of its ability to detect LN metastases. OSNA assay has also demonstrated a high diagnostic Regarding PCa, CK19 has been found not only in neo- accuracy for the detection of LN tumour burden in PCa, plastic tissue but also in basal and luminal cells of normal, but more studies are needed to confirm its validation. Cuadras et al. BMC Cancer (2022) 22:357 Page 9 of 10 Abbreviations 7. Briganti A, Chun FK-H, Salonia A, Suardi N, Gallina A, Da Pozzo LF, et al. CK19: Cytokeratin 19; ePLND: Extended pelvic lymph node dissection; FFPE: Complications and other surgical outcomes associated with extended Formalin-fixed paraffin-embedded; HNSCC: Head and neck squamous cell pelvic lymphadenectomy in men with localized prostate Cancer. Eur Urol. carcinomas; H&E: Haematoxylin and eosin; IHC: Immunohistochemistry; LN: 2006;50(5):1006–13. Lymph node; OSNA: One-step nucleic acid amplification; PCa: Prostate cancer; 8. Briganti A, Larcher A, Abdollah F, Capitanio U, Gallina A, Suardi N, et al. PSA: Prostate specific antigen; PRISMA: Preferred reporting items for system- Updated nomogram predicting lymph node invasion in patients with atic reviews and meta-analyses; QUADAS 2: Quality assessment of diagnostic prostate cancer undergoing extended pelvic lymph node dissection: accuracy studies 2; RT-PCR: Reverse transcription-polymerase chain reaction; the essential importance of percentage of positive cores. Eur Urol. SLN: Sentinel lymph node; TAB: Tumour allocation bias; TTL: Total tumour load. 2012;61(3):480–7. 9. Gandaglia G, Ploussard G, Valerio M, Mattei A, Fiori C, Fossati N, et al. Acknowledgements A novel nomogram to identify candidates for extended pelvic lymph Not applicable. node dissection among patients with clinically localized prostate Cancer diagnosed with magnetic resonance imaging-targeted and systematic Authors’ contributions biopsies. Eur Urol. 2019;75(3):506–14. JM participated in the evidence acquisition. JM, ET, and JP contributed to writ- 10. Lawrence WD. Association of Directors of anatomic and surgical pathol- ing the manuscript. All authors read and approved the final manuscript. ogy. ADASP recommendations for processing and reporting of lymph node specimens submitted for evaluation of metastatic disease. Virchows Funding Arch. 2001;439(5):601–3. The authors would like to thank SYSMEX for their insightful suggestions and 11. Schostak M, Krause H, Miller K, Schrader M, Kempkensteffen C, Koller- careful reading of the manuscript. Furthermore authors want to thank the mann J. Does the molecular staging in pelvic lymph nodes improve the funding support for the English adaptation and the Open Access fee. detection of relevant prostate cancer metastases? An assessment after 6years. BJU Int. 2007;99(6):1409–14. Availability of data and materials 12. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human All the data and materials analysed are included in the main paper. Further cytokeratins: patterns of expression in normal epithelia, tumours and data are available from the corresponding author on reasonable request. cultured cells. Cell. 1982;31(1):11–24. 13. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-medi- ated isothermal amplification using loop primers. Mol Cell Probes. Declarations 2002;16(3):223–9. 14. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Ethics approval and consent to participate et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. Not applicable. 2000;28(12):E63. 15. Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama Consent for publication F, et al. One-step nucleic acid amplification for intraoperative detection Not applicable. of lymph node metastasis in breast Cancer patients. Clin Cancer Res. 2007;13(16):4807–16. Competing interests 16. Fanfani F, Monterossi G, Di Meo ML, La Fera E, Dell’orto F, Gioè A, et al. The authors declare that they have no competing interests. Standard ultra-staging compared to one-step nucleic acid amplifica- tion for the detection of sentinel lymph node metastasis in endometrial Author details cancer patients: a retrospective cohort comparison. Int J Gynecol Cancer. 1 Urology Department, Vall d’Hebron University Hospital, Barcelona, Spain. 2020;30(3):372–7. 2 Pathology Department, Vall d’Hebron University Hospital, Barcelona, Spain. 3 17. Tranoulis A, Georgiou D, Yap J, Attard-Montalto S, Twigg J, Elattar A, et al. Surgery Department, Universitat Autònoma de Barcelona, Barcelona, Spain. The evolving role of one-step nucleic acid amplification (OSNA) for the intra-operative detection of lymph node metastases: a diagnostic accu- Received: 23 August 2021 Accepted: 28 January 2022 racy meta-analysis. Eur J Surg Oncol. 2021;47(6):1233–1243. 18. Perry N, Broeders M, de Wolf C, Törnberg S, Holland R, von Karsa L. European guidelines for quality assurance in breast cancer screen- ing and diagnosis. Fourth edition - summary document. Ann Oncol. 2008;19:614–22. References 19. Hiyoshi Y, Akiyoshi T, Fukunaga Y. The advantage of one-step nucleic 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global acid amplification for the diagnosis of lymph node metastasis in colo- cancer statistics 2018: Globocan estimates of incidence and mor- rectal cancer patients. Ann Gastroenterol Surg. 2021;5:60–6 Blackwell tality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. Publishing Ltd. 2018;68(6):394–424. 20. García-Alfonso P, García-Carbonero R, García-Foncillas J, Pérez-Segura P, 2. Bianchi L, Gandaglia G, Fossati N, Suardi N, Moschini M, Cucchiara V, et al. Salazar R, Vera R, et al. Update of the recommendations for the determi- Pelvic lymph node dissection in prostate Cancer: indications, extent and nation of biomarkers in colorectal carcinoma: National Consensus of the tailored approaches. Urol J. 2017;84(1):9–19. Spanish Society of Medical Oncology and the Spanish Society of Pathol- 3. Joniau S, Briganti A, Gontero P, Gandaglia G, Tosco L, Fieuws S, et al. ogy. Clin Transl Oncol. 2020;22(11):1976–91. Stratification of high-risk prostate Cancer into prognostic categories: a 21. Peg V, Espinosa-Bravo M, Vieites B, Vilardell F, Antúnez JR, De Salas MS, European multi-institutional study. Eur Urol. 2015;67(1):157–64. et al. Intraoperative molecular analysis of total tumour load in sentinel 4. Hövels AM, Heesakkers RAM, Adang EM, Jager GJ, Strum S, Hoogeveen lymph node: a new predictor of axillary status in early breast cancer YL, et al. The diagnostic accuracy of CT and MRI in the staging of pelvic patients. Breast Cancer Res Treat. 2013;139(1):87–93. lymph nodes in patients with prostate cancer: a meta-analysis. Clin 22. Peg V, Sansano I, Vieites B, Bernet L, Cano R, Córdoba A, et al. Role of total Radiol. 2008;63(4):387–95. tumour load of sentinel lymph node on survival in early breast cancer 5. Corfield J, Perera M, Bolton D, Lawrentschuk N. 68Ga-prostate specific patients. Breast. 2017;33:8–13. membrane antigen (PSMA) positron emission tomography (PET) for 23. Winter A, Engels S, Goos P, Süykers M-C, Henke R-P, Gerullis H, et al. Detec- primary staging of high-risk prostate cancer: a systematic review. World J tion of CK19 mRNA using one-step nucleic acid amplification (OSNA) in Urol. 2018;36(4):519–27. prostate Cancer: preliminary results. J Cancer. 2018;9(24):4611–7. 6. van Leeuwen PJ, Emmett L, Ho B, Delprado W, Ting F, Nguyen Q, et al. 24. Engels S, Brautmeier L, Reinhardt L, Wasylow C, Hasselmann F, Henke RP, Prospective evaluation of 68Gallium-prostate-specific membrane antigen et al. Evaluation of fast molecular detection of lymph node metastases in positron emission tomography/computed tomography for preoperative prostate cancer patients using one-step nucleic acid amplification (Osna). lymph node staging in prostate cancer. BJU Int. 2017;119(2):209–15. Cancers (Basel). 2021;13(5):1–12. Cuadras et al. BMC Cancer (2022) 22:357 Page 10 of 10 25. Shamseer L, Moher D, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. metastases using a one-step nucleic acid amplification (OSNA) assay in Preferred reporting items for systematic review and meta-analysis endometrial Cancer. Ann Surg Oncol. 2015;22(3):980–6. protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015 44. Fanfani F, Monterossi G, Ghizzoni V, Rossi ED, Dinoi G, Inzani F, et al. One- Jan;350:g7647. step nucleic acid amplification (OSNA): a fast molecular test based on 26. Huang X, Lin J, Demner-Fushman D. Evaluation of PICO as a knowl- CK19 mRNA concentration for assessment of lymph-nodes metastases in edge representation for clinical questions. AMIA Annu Symp Proc. early stage endometrial cancer. PLoS One. 2018;13(4):e0195877. 2006;2006:359–63. 45. Kosťun J, Pešta M, Sláma J, Slunéčko R, Vlasák P, Bouda J, et al. One-step 27. Whiting PF, Rutjes AWS, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, nucleic acid amplification vs ultrastaging in the detection of sentinel et al. QUADAS-2: a revised tool for the quality assessment of diagnostic lymph node metastasis in endometrial cancer patients. J Surg Oncol. accuracy studies. Ann Intern Med. 2011;155(8):529. 2019;119(3):361–9. 28. Feldman S, Krishnamurthy S, Gillanders W, Gittleman M, Beitsch PD, 46. Inoue M, Hiyama K, Nakabayashi K, Morii E, Minami M, Sawabata N, et al. Young PR, et al. A novel automated assay for the rapid identifica- An accurate and rapid detection of lymph node metastasis in non-small tion of metastatic breast carcinoma in sentinel lymph nodes. Cancer. cell lung cancer patients based on one-step nucleic acid amplification 2011;117(12):2599–607. assay. Lung Cancer. 2012;78(3):212–8. 29. Le Frère-Belda M-A, Bats A-S, Gillaizeau F, Poulet B, Clough KB, Nos C, et al. 47. Nakagawa K, Asamura H, Tsuta K, Nagai K, Yamada E, Ishii G, et al. The Diagnostic performance of one-step nucleic acid amplification for intra- novel one-step nucleic acid amplification (OSNA) assay for the diagnosis operative sentinel node metastasis detection in breast cancer patients. of lymph node metastasis in patients with non-small cell lung cancer Int J Cancer. 2012;130(10):2377–86. (NSCLC): results of a multicenter prospective study. Lung Cancer. 30. Vegué LB, Rojo F, Hardisson D, Iturriagagoitia AC, Panadés MJ, Velasco A, 2016;97:1–7. et al. Comparison of molecular analysis and histopathology for axillary 48. Escalante Pérez M, Hermida Romero MT, Otero Alén B, Álvarez Martínez lymph node staging in primary breast cancer: results of the B-CLOSER-I M, Fernández Prado R, de la Torre BM, et al. Detection of lymph node study. Diagn Mol Pathol. 2012;21(2):69–76. metastasis in lung cancer patients using a one-step nucleic acid 31. Wang Y, Ou-yang T, Wu J, Liu Y, Cao X, Sun X, et al. Comparative study amplification assay: a single-Centre prospective study. J Transl Med. of one-step nucleic acid amplification assay, frozen section, and touch 2019;17(1):233. imprint cytology for intraoperative assessment of breast sentinel lymph 49. Goda H, Nakashiro K, Oka R, Tanaka H, Wakisaka H, Hato N, et al. One-step node in Chinese patients. Cancer Sci. 2012;103(11):1989–93. nucleic acid amplification for detecting lymph node metastasis of head 32. Pathmanathan N, Renthawa J, French JR, Edstrom-Elder E, Hall G, Mahajan and neck squamous cell carcinoma. Oral Oncol. 2012;48(10):958–63. H, et al. Intraoperative sentinel lymph node assessment in breast cancer: 50. del Carmen S, Gatius S, Franch-Arcas G, Baena JA, Gonzalez O, Zafon C, a comparison of rapid diagnostic method based on CK19 mRNA expres- et al. Concordance study between one-step nucleic acid amplification sion and imprint cytology. ANZ J Surg. 2014;84(10):730–4. and morphologic techniques to detect lymph node metastasis in papil- 33. Banerjee SM, Michalopoulos NV, Williams NR, Davidson T, El Sheikh S, lary carcinoma of the thyroid. Hum Pathol. 2016;48:132–41. McDermott N, et al. Detailed evaluation of one step nucleic acid (OSNA) 51. Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. molecular assay for intra-operative diagnosis of sentinel lymph node Histopathology. 2002;40(5):403–39. metastasis and prediction of non-sentinel nodal involvement: experience 52. Bernet L, Piñero A, Vidal-Sicart S, Peg V, Giménez J, Algara M, et al. from a London teaching hospital. Breast. 2014;23(4):378–84. Consenso sobre la biopsia selectiva del ganglio centinela en el cáncer de 34. Li D, Xu X, Chen J, Chen J, Yang B, Yang W, et al. Utility of one-step nucleic mama. Revisión 2013 de la Sociedad Española de Senología y Patología acid amplification (OSNA) assay in detecting breast cancer metasta- Mamaria. Rev Esp Patol. 2014;27(1):43–53. ses of sentinel lymph nodes in a Chinese population. Breast Cancer. 53. Oncoguía SEGO. Cáncer infiltrante de mama. Guías de práctica clínica en 2015;22(2):135–40. cáncer ginecológico y mamario. Publicaciones SEGO, Junio; 2017. 35. Croner RS, Schellerer V, Demund H, Schildberg C, Papadopulos T, 54. Rakislova N, Montironi C, Aldecoa I, Fernandez E, Bombi JA, Jimeno Naschberger E, et al. One step nucleic acid amplification (OSNA) - a new M, et al. Lymph node pooling: a feasible and efficient method of method for lymph node staging in colorectal carcinomas. J Transl Med. lymph node molecular staging in colorectal carcinoma. J Transl Med. 2010;8:83. 2017;15(1):14. 36. Yamamoto H, Sekimoto M, Oya M, Yamamoto N, Konishi F, Sasaki J, et al. 55. Archilla I, Díaz-Mercedes S, Aguirre JJ, Tarragona J, Machado I, Rodrigo OSNA-based novel molecular testing for lymph node metastases in colo- MT, et al. Lymph node tumour burden correlates with tumour budding rectal Cancer patients: results from a multicenter clinical performance and poorly differentiated clusters: a new prognostic factor in colorectal study in Japan. Ann Surg Oncol. 2011;18(7):1891–8. carcinoma? Clin Transl Gastroenterol. 2021;12(3):e00303. 37. Güller U, Zettl A, Worni M, Langer I, Cabalzar-Wondberg D, Viehl CT, et al. 56. Bettington M, Lakhani SR, Ung OA. Is the one-step nucleic acid amplifica- Molecular investigation of lymph nodes in colon cancer patients using tion assay better for intra-operative assessment of breast sentinel nodes? one-step nucleic acid amplification (OSNA): a new road to better staging? ANZ J Surg. 2014;84(10):725–9. Cancer. 2012;118(24):6039–45. 57. Peehl DM, Sellers RG, McNeal JE. Keratin 19 in the adult human prostate: 38. Yamamoto H, Tomita N, Inomata M, Furuhata T, Miyake Y, Noura S, et al. tissue and cell culture studies. Cell Tissue Res. 1996;285(1):171–6. OSNA-assisted molecular staging in colorectal Cancer: a prospective 58. Mottet N, Van Den Bergh RCN, Briers E, Van Den Broeck T, Cumberbatch multicenter trial in Japan. Ann Surg Oncol. 2016;23(2):391–6. MG, De Santis M, et al. EAU-EANM-ESTRO-ESUR-SIOG guidelines on pros- 39. Yaguchi Y, Sugasawa H, Tsujimoto H, Takata H, Nakabayashi K, Ichikura tate cancer-2020 update. Part 1: screening, diagnosis, and local treatment T, et al. One-step nucleic acid amplification (OSNA) for the applica- with curative intent. 2020. tion of sentinel node concept in gastric Cancer. Ann Surg Oncol. 2011;18(8):2289–96. 40. Kumagai K, Yamamoto N, Miyashiro I, Tomita Y, Katai H, Kushima R, et al. Publisher’s Note Multicenter study evaluating the clinical performance of the OSNA assay Springer Nature remains neutral with regard to jurisdictional claims in pub- for the molecular detection of lymph node metastases in gastric cancer lished maps and institutional affiliations. patients. Gastric Cancer. 2014;17(2):273–80. 41. Shimada A, Takeuchi H, Nishi T, Mayanagi S, Fukuda K, Suda K, et al. Utility of the one-step nucleic acid amplification assay in sentinel node map- ping for early gastric cancer patients. Gastric Cancer. 2020;23(3):418–425. 42. Okamoto S, Niikura H, Nakabayashi K, Hiyama K, Matoda M, Takeshima N, et al. Detection of sentinel lymph node metastases in cervical cancer: assessment of KRT19 mRNA in the one-step nucleic acid amplification (OSNA) method. Gynecol Oncol. 2013;130(3):530–6. 43. Nagai T, Niikura H, Okamoto S, Nakabayashi K, Matoda M, Utsunomiya H, et al. A new diagnostic method for rapid detection of lymph node