BACKGROUND:
This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability.
METHODS:
We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria.
RESULTS:
In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06-6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69-0.92) was achieved. For TTL >1.9 × 105 copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases.
CONCLUSION:
When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection.info:eu-repo/semantics/publishedVersio

Araújo, A

Figueiredo, J

Lopes, P

Martins, M

Nabais, C

Repository of the Hospital Center of Central Lisbon

Total Tumor Load Assessed by One-Step Nucleic Acid Amplification Assay as an Intraoperative Predictor for Non-Sentinel Lymph Node Metastasis in Breast Cancer

Repositório do Centro Hospitalar de Lisboa Central, EPE

lable at ScienceDirectThe Breast 32 (2017) 33e36Contents lists avaiThe Breastjournal homepage: www.elsevier .com/brstOriginal articleTotal tumor load assessed by one-step nucleic acid ampliﬁcation assayas an intraoperative predictor for non-sentinel lymph node metastasisin breast cancerCelso Nabais a, *, Joana Figueiredo a, Paulina Lopes a, Manuela Martins b, Antonio Araújo aa Department of Surgery, Centro Hospitalar Lisboa Central, Portugalb Department of Pathology, Centro Hospitalar Lisboa Central, Portugala r t i c l e i n f oArticle history:Received 15 August 2016Received in revised form1 October 2016Accepted 19 December 2016Keywords:Breast cancerOne-step nucleic acid ampliﬁcationSentinel lymph nodeNon-sentinel lymph node metastasisAxillary lymph node dissectionTotal tumor load* Corresponding author. Department of Surgery, CenRua Jose Antonio Serrano, 1150-199, Lisboa, Portugal.E-mail address: celso.nabais@gmail.com (C. Nabaihttp://dx.doi.org/10.1016/j.breast.2016.12.0110960-9776/© 2016 Elsevier Ltd. All rights reserved.a b s t r a c tBackground: This study aimed to determine the relationship between CK19 mRNA copy number insentinel lymph nodes (SLN) assessed by one-step nucleic acid ampliﬁcation (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load(TTL) obtained by OSNA technique was also constructed to evaluate its predictability.Methods: We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulﬁll theinclusion criteria.Results: In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection.Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictivefactors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR2.67; 95% CI 1.06e6.70; P ¼ 0.036). The ROC curve for the model using TTL alone was obtained and anAUC of 0.805 (95% CI 0.69e0.92) was achieved. For TTL >1.9  105 copies/mL we got 73.3% sensitivity,74.4% speciﬁcity and 88.9% negative predictive value to predict NSLN metastases.Conclusion: When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intra-operatively. This prediction tool could help in decision for axillary lymph node dissection.© 2016 Elsevier Ltd. All rights reserved.1. IntroductionSentinel lymph node (SLN) biopsy have become the standardtechnique for determining axillary nodal involvement in patientswith early-stage breast cancer who are clinically negative.Identifying the SLN as non-metastatic spares unnecessary axil-lary lymph node dissection (ALND) and therefore decreases thechances of signiﬁcant co-morbidities [1].Intraoperative diagnosis of positive SLN can allow ALND in thesame surgical procedure when criteria are present, thus avoiding asecond surgery to treat the axilla and decreasing the patient'sassociated discomfort and institutional costs [2,3].The one-step nucleic acid ampliﬁcation (OSNA, Sysmex, Kobe,Japan) assay is a molecular method that measures the quantity ofcytokeratin (CK)-19mRNA (aduct epithelial cellmarker that is highlytro Hospitalar Lisboa Central,s).expressed in more than 95% of breast cancers) in axillary lymphnodes [4]. Cutoff values were deﬁned to classify macrometastases(more than 5000 copies/mL),micrometastases (250e5000 copies/mL)and negative nodes (fewer than 250 copies/mL) [5].Combined analysis of nine studies that compared OSNA withhistopathology demonstrated high concordance between bothmethods (96%) and reported high sensitivity, speciﬁcity and nega-tive predictive value for OSNA [2]. As the OSNA assay is essentiallyan automated procedure, it has clear advantages in standardization,reproducibility and objectivity.There is so far no clear consensus on how to approach ALND.Several studies have identiﬁed predictors of metastases to non-sentinel lymph nodes (NSLNs), to select patients who can bespared ALND, and different nomograms have been proposed toselect patients who would not beneﬁt from ALND.Recently, the American College of Surgeons Oncology Group(ACOSOG) Z0011 trial deﬁned a select cohort of patients withpositive SLNs in whom a complete ALND may be safely omitted [6].However, many patients still require prediction of non-SLNmetastases.C. Nabais et al. / The Breast 32 (2017) 33e3634With the increasing use of OSNA, different groups have startedto study the relationship between CK19 mRNA copy number andthe NSLN metastization.Ohi et al. (2012) and Osako et al. (2013) demonstrated that theNSLN macrometastatic rate increased in proportion to CK19 mRNAcopy numbers [7,8]; Ohi et al. veriﬁed that the CK19 mRNA copynumber in SLN is the most important predictive factor of NSLNmetastases, and that higher copy numbers are strongly associatedwith four or more axillary lymph node metastases [7].Other groups also evaluated the correlation between the totaltumor load (TTL) in SLN and additional NSLN metastases [9,10].Banerjee et al. (2014), in a small subgroup of 45 women who hadundergone ALND, found that using the CK19 mRNA copy numberalone resulted in an AUC of 0.828, which indicates that OSNA ismore useful than nomograms in predicting the risk of NSLNmetastasis [2].This study aimed to determine the relationship between CK19mRNA copy numbers and subsequent NSLN metastization, and todetermine the TTL intraoperatively as a threshold above whichmetastases are expected, to support surgeons' oncological de-cisions regarding the need to perform ALND.2. Materials and methodsThis observational retrospective study was conducted betweenOctober 2010 and December 2014, and initially enrolled 598women with invasive breast cancers. Inclusion criteria were pa-tients whose disease had been assessed clinically and ultrasono-graphically as node-negative and at tumor stage cT1e3, and whohad undergone intraoperative SLN evaluation by OSNA. Weexcluded patients who had received neoadjuvant treatment, whosebiopsies showed CK19-negative tumors, or those who did not un-dergo ALND. Of the 598 patients, 88 had positive SLNs. Of these 88,61 had been analyzed by OSNA, three of whom were excludedbecause they had not undergone ALND. Finally, 58 valid patientswere studied. Data collected included age, tumor size, grade, his-tological subtype, estrogen and progesterone receptor status, HER2status, Ki67, lymphovascular invasion, multifocality, total numberTable 1Patient's characteristics and comparison of negative versus positive NSLN.Negative NSLN (n ¼ 43Age e years (mean ± SD) 57.10 ± 11.09Histologic typeInvasive ductal carcinoma 35Invasive þ ductal carcinoma in situ 3Invasive lobular carcinoma 5Invasive papillary carcinoma 0Tumor size (mean in mm ± SD) 20.65 ± 7.06Lymphovascular invasionYes 20No 20ERPositive 36Negative 6PRPositive 29Negative 13HER2Positive 7Negative 30SLN number (mean ± SD) 2.23 ± 1.15SLN macrometastases (mean ± SD) 1.19 ± 0.55NSLN resected (mean ± SD) 11.86 ± 6.11TTL - log (mean ± SD) 4.65 ± 0.84ER: estrogen receptors; PR: progesterone receptors; HER2: human epidermal growth fatotal tumor load.of SLNs and non-SLNs, type of surgery, the number of positive andnegative non-SLNs, and CK19 mRNA copies.SLNs were detected using radioisotopes and blue dye, and sentfor pathological analysis. When macrometastases were foundduring intraoperative evaluation, patients underwent level IIALNDs. Depending on patient and tumor characteristics, lumpec-tomies or mastectomies were also performed.OSNA evaluations were completed for the isolated SLNs. TheOSNA assay procedure has already been described in detail [4]. Theanalysis result included the number of CK19 mRNA copies per mL.These copy numbers were used semi-quantitatively to characterizenode involvement; those with <250 copies/mL were considerednon-metastatic, 250e5000 copies/mL as having micrometastases,and >5000 copies/mL as having macrometastases.NSLNs obtained from ALND were studied after being processedby histopathologic methods. Immunohistochemical staining wasnot used.2.1. Statistical analysisData were evaluated descriptively, with frequencies used forcategorical variables and medians for continuous variables. Chi-square and ANOVA tests were used to compare positive andnegative NSLNs. We conducted univariate and multivariate ana-lyses, from which non-signiﬁcant variables (P > 0.05) were drop-ped. Logistic regression was used to assess the capacity of thestudied variables to identify positive NSLNs.The TTL variable was studied using area under the receiveroperating characteristic (ROC) curve (AUC), after log transformationto avoid nonlinearities. The statistical analyses were carried out inSPSS 20.0 for Windows and MedCalc 15.8.3. Results3.1. Patients' characteristicsOf the 58 patients with positive SLNs who were analyzed byOSNA in this study, 15 (25.86%) were found to have positive nodes) Positive NSLN (n ¼ 15) P value57.77 ± 12.68 0.7170.3711211125.93 ± 10.79 0.0350.469850.4401410.0611410.6992122.60 ± 1.06 0.2821.80 ± 0.68 0.00112.80 ± 5.36 0.5995.54 ± 0.71 0.000ctor receptor 2; SLN: sentinel lymph nodes; NSLN: non-sentinel lymph nodes; TTL:Table 2Univariate and multivariate analyses of NSLN macrometastases.OR (95% CI)UnivariatePUnivariateOR (95% CI)MultivariatePMultivariateAge (years) 0.99 (0.94e1.05) 0.711Tumor size (mm) 1.08 (1.00e1.16) 0.047 1.06 (0.98e1.15) 0.168Log TTL (copies/mL) 3.56 (1.57e8.09) 0.002 2.67 (1.06e6.70) 0.036SLN macrometastases 4.64 (1.55e13.92) 0.006 2.63 (0.94e7.36) 0.066Histologic typeInvasive ductal carcinoma 1 0.974Invasive þ ductal carcinoma in situ 0.97 (0.09e10.26)Invasive lobular carcinoma 0.58 (0.06e5.51)Invasive papillary carcinoma eHistological tumor gradeI 1 0.069II 0.15 (0.02e1.05)III 1.14 (0.18e7.28)Lymphovascular invasion (yes vs no) 1.60 (0.45e5.74) 0.471ER (positive vs negative) 2.33 (0.26e21.17) 0.451PR (positive vs negative) 6.28 (0.75e52.90) 0.091HER2 (positive vs negative) 0.71 (0.13e3.94) 0.699Multifocality (yes vs no) 2.78 (0.53e14.48) 0.226ER: estrogen receptors; PR: progesterone receptors; HER2: human epidermal growth factor receptor 2; SLN: sentinel lymph nodes; TTL: total tumor load.Fig. 1. ROC Curve using TTL variable as predictor of NSLN metastases.C. Nabais et al. / The Breast 32 (2017) 33e36 35in ALND. The eight patients had a mean age of 56.7 years, meantumor size of 21.79 mm, mean SLN number of 2.31, and 1.36 mac-rometastases. The mean TTL was 5.86  105 copies/mL. Moredetailed patients' characteristics, stratiﬁed by positive or negativeNSLN, are shown in Table 1.3.2. Univariate and multivariate analysesIn univariate analysis, we considered age, tumor size, TTL, SLNmacrometastases, histologic type, histological tumor grade, lym-phovascular invasion, ER, PR, HER2 and multifocality; and foundtumor size, TTL and number of SLN macrometastases to be pre-dictive factors of NSLN metastases (P < 0.05; Table 2).In multivariate analysis, TTL was the only independent predictorof NSLN metastases (odds ratio [OR]: 2.67; 95% conﬁdence interval[CI]: 1.06e6.70; P ¼ 0.036; Table 2). Both tumor size and number ofSLN macrometastases were signiﬁcantly associated with additionalNSLN metastases in univariate analysis, but not in multivariateanalysis (OR: 1.06; 95% CI: 0.98e1-15; P ¼ 0.168 and OR: 2.63; 95%CI: 0.94e7.36; P ¼ 0.066, respectively).Intraoperative TTL assessment by OSNA was further studied byROC curve. The AUC of TTL/log TTL was 0.805 (95% CI 0.69e0.92;P < 0.05; Fig. 1). We then used the ROC curve to choose a cutoffpoint for TTL at log TTL >5.28 or TTL >1.9  105 copies/mL, whichgave 73.3% sensitivity, 74.4% speciﬁcity, and 88.9% negative pre-dictive value for NSLN metastases. In this sample there were 15patients with NSLN metastases, 11 of whom had TTL >1.9  105copies/mL.4. DiscussionSLNs are the ﬁrst axillary nodes to receive lymphatic ﬂow fromprimary tumors and themost likely to harbor tumor cells. If SLN hasno metastases, ALND can be safely avoided [1]. However, if the SLNis positive for macrometastases, ALND is the standard procedure,even though more than half of these patients have no NSLN me-tastases. In our study, only 25.86% of patients with positive SLNshad NSLN metastases.Several prediction models for NSLN metastases in patients withpositive SLNs have been developed, but most are based on post-operative histological ﬁndings, and are therefore not helpful forintraoperative decision making.OSNA assay is a molecular-based intraoperative detectionmethod for SLN metastases that has several advantages over frozensections; it is a fast, standardized technique in which the entirelymph node is examined and provides quantitative results [2].In this study, univariate analysis showed that tumor size,number of SLN macrometastases and CK19 mRNA copies (TTL)determined by OSNA assay were signiﬁcantly associated with non-SLN metastases. These results are in line with other studies [9,10].However, in multivariate analysis, TTL was the only variable with asigniﬁcant association that could be used intraoperatively.Reportedly, TTL can be used as a predictor for non-SLN metas-tases [9,10]. We studied the prediction model with TTL using theROC curve, which had good discrimination acuity (AUC ¼ 0.805) inthis group of patients. At a TTL cutoff of >1.9  105 copies/mL, therewas 73.3% sensitivity and 74.4% speciﬁcity in predicting NSLN me-tastases. This cutoff was calculated considering a low false positiverate to avoid hypothetically unnecessary ALND in post Z0011 era.Espinosa-Bravo et al. (2013) also reported similar results [9].After the results of the ACOSOG Z0011 trial were reported, thestandard practice of ALND in the presence of positive SLNs wasC. Nabais et al. / The Breast 32 (2017) 33e3636questioned. This trial indicated that ALND has no signiﬁcant impacton either disease-free or overall survival of patients with SLN me-tastases, but these results are only applicable to patients who metthe study inclusion criteria (fewer than three SLN metastases,treatment with breast-conserving surgery, radiation therapy andsystemic therapy) [6].In patients for whom the results of the Z0011 are not applicable,prediction models for NSLN metastases are still helpful for ALNDsurgical decisions. The current predictor model using TTL can beintraoperatively valuable, as a simple, fast and accurate method ofassessing the probability of NSLN.Our study has the limitations of being retrospective with a smallsample size. The clinical implications of TTL in how we surgicallymanage patients are therefore limited. Larger prospective studiesare needed to determine the prognostic implications.Ethical approvalEthical approval was not required for this review.Funding sourceThis research did not receive any speciﬁc grant from fundingagencies in the public, commercial, or not-for-proﬁt sectors.Conﬂict of interest statementNone declared.AcknowledgementsWe would like to thank Ana Correia, Ine^s Moruj~ao and CarlosVitorino for providing helpful support and suggestions during theresearch.References[1] Veronesi U, Paganelli G, Galimberti V, Viale G, Zurrida S, Bedoni M, et al.Sentinel-node biopsy to avoid axillary dissection in breast cancer with clini-cally negative lymph-nodes. Lancet Lond Engl 1997 Jun 28;349(9069):1864e7.[2] Banerjee SM, Michalopoulos NV, Williams NR, Davidson T, El Sheikh S,McDermott N, et al. Detailed evaluation of one step nucleic acid (OSNA)molecular assay for intra-operative diagnosis of sentinel lymph node metas-tasis and prediction of non-sentinel nodal involvement: experience from aLondon Teaching Hospital. Breast 2014 Aug;23(4):378e84.[3] Heilmann T, Mathiak M, Hofmann J, Mundhenke C, van Mackelenbergh M,Alkatout I, et al. Intra-operative use of one-step nucleic acid ampliﬁcation(OSNA) for detection of the tumor load of sentinel lymph nodes in breastcancer patients. J Cancer Res Clin Oncol 2013 Oct;139(10):1649e55.[4] Chaudhry A, Williams S, Cook J, Jenkins M, Sohail M, Calder C, et al. The real-time intra-operative evaluation of sentinel lymph nodes in breast cancerpatients using One Step Nucleic Acid Ampliﬁcation (OSNA) and implicationsfor clinical decision-making. Eur J Surg Oncol EJSO 2014 Feb;40(2):150e7.[5] Tiernan JP, Verghese ET, Nair A, Pathak S, Kim B, White J, et al. Systematicreview and meta-analysis of cytokeratin 19-based one-step nucleic acidampliﬁcation versus histopathology for sentinel lymph node assessment inbreast cancer: sentinel lymph node assessment in breast cancer. Br J Surg2014 Mar;101(4):298e306.[6] Lyman GH, Temin S, Edge SB, Newman LA, Turner RR, Weaver DL, et al.Sentinel lymph node biopsy for patients with early-stage breast cancer:american society of clinical Oncology clinical practice guideline update. J ClinOncol 2014 May 1;32(13):1365e83.[7] Ohi Y, Umekita Y, Sagara Y, Rai Y, Yotsumoto D, Matsukata A, et al. Wholesentinel lymph node analysis by a molecular assay predicts axillary nodestatus in breast cancer. Br J Cancer 2012 Oct 9;107(8):1239e43.[8] Osako T, Iwase T, Kimura K, Horii R, Akiyama F. Sentinel node tumour burdenquantiﬁed based on cytokeratin 19 mRNA copy number predicts non-sentinelnode metastases in breast cancer: molecular whole-node analysis of allremoved nodes. 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Total Tumor Load Assessed by One-Step Nucleic Acid Amplification Assay as an Intraoperative Predictor for Non-Sentinel Lymph Node Metastasis in Breast Cancer

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