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Tumoral load quantification of positive sentinel lymph nodes in breast cancer to predict more than two involved nodes
The Breast 23 (2014) 859e864
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The Breast journal homepage: www.elsevier.com/brst
Original article
Tumoral load quantification of positive sentinel lymph nodes in breast cancer to predict more than two involved nodes ~ ero-Madrona a, *, Guadalupe Ruiz-Merino b, Laia Bernet c, Antonio Pin ~ a Miguel-Martínez d, Francisco Vicente-García e, María A. Viguri-Díaz f, Begon nez-Climent g Julia Gime a
Department of Surgery, “Virgen de la Arrixaca” University Hospital, 30120 Murcia, Spain n, 9, 1ª Planta, 30120 Murcia, Spain Department of Statistics, FFIS-IMIB, Luis Fontes Paga tiva, Crtra de Xa tiva a Silla, Km 1, Valencia, Spain Department of Pathology, Hospital of Xa d Department of Nuclear Medicine, University Hospital, Hijos de Santiago Rodriguez, 16, 09002 Burgos, Spain e Department of Surgery, Complejo Hospitalario de Navarra, La Arboleda, 4, Cizur Menor, 31190 Pamplona, Spain f Department of Pathology, University Hospital Txagorritxu, Jose Atxotegui s/n, 01009 Vitoria, Spain g n Ba guena, 8, 46009 Valencia, Spain n Instituto Valenciano de Oncología, Prof. Beltra Department of Surgery, Fundacio b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 April 2014 Received in revised form 9 September 2014 Accepted 12 September 2014 Available online 3 October 2014
Aim: One-Step Nucleic Acid Amplification (OSNA) can detect isolated tumour loads in axillary lymph nodes of breast cancer patients. We investigated the predictability of the non-sentinel lymph node (SLN) metastatic involvement (MI) based on the OSNA SLN assessment in surgical invasive breast cancer. Methods: We studied surgical breast invasive carcinoma patients, not taking neoadjuvant chemotherapy, having SLN positive by OSNA and having received axillary lymphadenectomy. Age, basic histopathological, immunohistochemical, SLN biopsy and lymphadenectomy data were compared between patients with or without MI of more than 2 non-SLN in both univariate and multivariate analyses. The discriminating capacity of the multivariate model was characterized by the ROC AUC. Results: 726 patients from 23 centers in Spain aged 55.3 ± 12.2 years were analysed. The univariate analysis comparing patients with or without MI of more than 2 non-SLN detected statistically significant differences in primary tumour size, multifocality, presence of lymphovascular infiltration, positive proliferation index with ki67, immunophenotype and logTTL (Tumour Total Load). The multivariate logistic analyses (OR (95% CI)) confirmed multifocality (2.16 (1.13e4.13), p ¼ 0.019), lymphovascular infiltration (4.36 (2.43e7.82), p < 0.001) and logTTL (1.22 (1.10e1.35), p < 0.001) as independent predictors, and exhibit an AUC (95% CI) of 0.78 (0.72e0.83) with an overall fit (HosmereLemeshow test) of 0.359. A change in the slope of both sensitivity and specificity is observed at about 10,000 copies/mL, without relevant changes in the Negative Predictive Values. Conclusions: Using OSNA technique, the MI of more than 2 non-SLN can be reliably predicted. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Breast neoplasms Intraoperative procedures Keratin 19 mRNA copy number One-step nucleic acid amplification assay Sentinel lymph-node biopsy
Introduction In the last twenty years, the election of the axillary locoregional treatment for breast cancer has become a key decision that can determine global and disease-free survival [1]. Studies initiated in
* Corresponding author. Tel.: þ34 968369677; fax: þ34 968395537. E-mail addresses: [email protected], [email protected] ~ ero-Madrona), [email protected] (G. Ruiz-Merino), [email protected] (A. Pin (L. Bernet), [email protected] (B. Miguel-Martínez), [email protected] (F. VicenteGarcía), [email protected] (M.A. Viguri-Díaz), julia. nez-Climent). [email protected] (J. Gime http://dx.doi.org/10.1016/j.breast.2014.09.005 0960-9776/© 2014 Elsevier Ltd. All rights reserved.
the 1980s suggested that the Axillary Lymph Node Dissection (ALND) entails an unacceptable risk of serious complications [2,3]. With the objective of benefit maximization and risk minimization, the standard ALND was reconsidered, and the Sentinel Lymph Node Dissection (SLND) technique was developed as a more precise technique in the staging of tumour draining axillary nodes, with less morbidity than ALND [4]. Nowadays, the SLND is generally accepted as the sole management for patients whose sentinel lymph nodes (SLNs) are unaffected according to histology or OSNA (One-Step Nucleic Acid Amplification), while ALND remains the standard of care for patients whose SLNs contain metastases [5]. In any case, choice of treatment implies other problems, and at
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present there is a need for further treatment optimization and standardization, so as to avoid unnecessary risks for patients. The intraoperative selective biopsy of SLN has represented a significant increase in the detection of micrometastases and submicrometastases as compared with previous periods [6,7]. Recent studies, both retrospective [8] and prospective [9,10], suggest the possibility of not carrying out ALND when only micrometastases are found in the SLN, especially in certain subgroups of good prognosis [11], or even in the case of macroscopic limited nodal disease [12]. More in detail, the results of the ACOSOG Z0011 study suggested that it could be possible to prevent ALND, even when the macroscopic nodal disease affected up to two SLN [12]. Thus, the interest would be to characterise which cases would be less probably affected by axillary disease beyond the two SLN, and more specifically in the case of a positive selective biopsy of SLN with an adequate negative predictive value (NPV), useful to predict their probability of appearance. In this context, advances in diagnostic technology based on molecular methods for the analysis of SLN might be essential to improve therapeutic management. OSNA is not able to detect isolate tumoral cells but only micro and macrometastases load through detection of mRNA CK19 expression in the node [13,14]. The OSNA method has shown a 96% concordance rate with detailed histopathology complemented with immunohistochemistry, a low false-negative rate, and a very low false-positive rate, what makes this technique suitable for the intraoperative evaluation of SLN [15]. On the other hand, intraoperative molecular analysis for SLN metastases using the OSNA method shortens admission time and the duration of surgery, and saves 439.67 V per patient in Spain [16]. It is tempting to change the gold standard for comparisons between methods, and when this is done, histology seems to come out as a weaker test for the identification of metastases. In any case, despite the advantage of detecting even very limited nodal involvement, the implications of this high sensitivity in deciding the convenience of ALND, or its magnitude, remain unclear. While some studies have suggested not to perform ALND in case of a low metastatic involvement (MI) rate of non-SLN [17e19], others have pointed out that this may occur in more than 20% of cases, and therefore they do not recommend to avoid lymphadenectomy [20e24]. Nevertheless, the sensitivity of the OSNA method clearly represents an opportunity for a fine assessment of the tumour load in axillary nodes, to determine a threshold for avoiding lymphadenectomy, and also to guide radiotherapy so as to reduce its associated morbidities [25e27]. The aim of the present work was to characterize the relationship between the tumour load in positive/s SLN, using OSNA method, and the presence of disease in two or more additional non-SLN in breast cancer patients, so as to establish a threshold to optimize the prediction of non-SLN MI in order to avoid ALND.
Methods Study design We conducted a multicenter observational study in patients with invasive breast carcinoma subjected to a selective SLN biopsy.
Setting The study was conducted in Breast Surgery Units from 23 Spanish hospitals. The study had been previously approved by the Ethics Committees of the participating centers. Medical records provided the necessary data for this study.
Participants We included all patients with a positive SLN biopsy according to the OSNA technique, that later underwent an ALND of at least 10 lymph nodes. We excluded patients with negative SLN, those that had received neoadjuvant chemotherapy, or that had SLN assessed with a method different from OSNA, or when the histopathological assessment of isolated lymph nodes from the ALND was other than usual (halved in two slices). SLN and ALND evaluation The Sentinel Lymph Node Biopsy was performed in the participating centres following the recommendations of the consensus document from the Senology and Mammary Pathology Spanish Society [28]. The OSNA assay was performed for the isolated sentinel lymph nodes analysis. The OSNA assay for lymph nodes has been previously described in detail [29]. Briefly, the number of mRNACK19 copies per ml in the measurement sample was calculated; these copy numbers were used as basis to determine the result. The cutoffs for negative/positive and micro-/macrometastases were set at 250 and 5000 copies/ml, respectively. The axillary lymph nodes from the lymphadenectomy were studied following conventional protocol, that implies isolation of all the nodes, and each one was processed by bipartition and histopathological study of one 4 m section of every piece from both sections. Immunohistochemistry for cytokeratine (AE1/AE3) was used only in doubtful cases. Variables and definitions For each patient included in the study, we recorded the number of biopsied SLNs (as selected by the surgeon intraoperatively), the total tumour load (TTL) from SLNs (mRNA CK19 copies/mL), the number of nodes isolated in ALND, and the number of those that were affected as per histopathological assessment. Cases were pooled for analysis in those patients with more than two affected nodes (following the lymphadenectomy criteria in recent series [12]). Other variables recorded were: patient's age, primary tumour size, histological type, histological grade (according to ScarffBloom-Richardson [30]), lymphovascular infiltration, bilaterality and immunophenotype classification (expression of oestrogenic and progesterone receptors, considering more than 10% dyed cells as positive, overexpression of Her-2-neu, considering þþþ and þþ with positive FISH as positive, and the ki67, considering>14% dyed cells as positive). Luminal A immunophenotype was described as ER and PR expression with low ki67 figures; Luminal B, when there was ER and PR expression with ki67 higher than 14%; Luminal BeH, when Her2neu overexpression was added to Luminal B profile; Her2neu was defined as Her2neu overexpression without ER or PR expression; and triple negative profile, when there was neither ER or PR expression or Her2. Statistical methods The sample size was determined by the availability of eligible cases in the participating centers during the study period, and no formal sample size was estimated. As a first step, a univariate analysis was conducted to compare study variables in patients with or without MI of more than 2 nonSLN, using the Student t-test, the Pearson chi-square test, or the Fisher's exact test as suitable. Then, a multivariate logistic
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regression model was built with those variables that showed significant results in the previous univariate analysis. A stepwise selection procedure was used to select the model predictors, with p < 0.05 in Wald tests as entry and retain criteria. We characterised the discriminating ability of the resulting model, and of some alternative reduced models, by plotting the corresponding ROC curves and computing the area under the curves (AUC). The data are described by means and standard deviations or counts and percentages, as suitable. Results from statistical tests were considered significant if two-sided p < 0.05. The statistical analysis was carried out with SPSS v.18.0.
SLN nodes were isolated in ALND (n ¼ 34), if they received neoadjuvant chemotherapy (n ¼ 16), had SLN assessed with a method different from OSNA, or had non-conventional histopathologic assessment of non-SLN. The total number of patients included in the analysis was 726.
Results
Univariate analysis
Participants
The univariate analysis comparing patients with or without MI of more than 2 non-SLN detected statistically significant differences in the primary tumour size, multifocality, presence of lymphovascular infiltration, positive proliferation index with ki67, luminal B immunophenotype, and (log) TTL (see Table 2).
From 2000 to 2012, 797 patients from 23 centers with a diagnosis of invasive breast carcinoma were operated, and sentinel lymph nodes biopsied. Patients were excluded if less than 10 non-
Descriptive data The age of patients was 55.3 ± 12.2 years (range: 28e89), and the size of the primary tumour was of 20.8 ± 13.0 mm (range: 1e150). Patients' characteristics are shown in Table 1.
Multivariate analysis Table 1 Patient's characteristics. Data are expressed as mean (SD) or n (%) as appropriate. Age (years) Hystological type IDC ILC C mixed Size (mm) Focality Unifocal Multifocal Not available Bilaterality Differentiation Good Moderate Scarce Not available Lymphovascular infiltration Yes No Not available ER Positive Negative Not available PR Positive Negative Not available HER2NEU Positive Negative Not available Ki67 Positive Negative Not available Immunophenotype Luminal A Luminal B Luminal BH HER Triple negative Not available Number of isolated SLN Number of affected SLN Number of lymphadenectomy isolated nodes Involved nodes in ALND
55.28 (12.20) 655 63 8 20.85
(90.2%) (8.7%) (1.1%) (13.01)
592 131 3 8
(81.5%) (18.1%) (0.4%) (1.1%)
179 372 145 30
(24.6%) (51.3%) (20.0%) (4.1%)
211 (29.0%) 365 (50.3%) 150 (20.7%) 547 (75.3%) 49 (6.8%) 130 (17.9%) 460 (63.3%) 132 (18.2%) 134 (18.5%) 61 (8.4%) 517 (71.2%) 148 (20.3%) 318 (43.8%) 262 (36.1%) 146 (20.1%) 238 262 30 15 33 148 1.73 1.09 17.30 1.29
(32.8%) (36.1%) (4.1%) (2.0%) (4.5%) (20.5%) (0.846) (0.293) (5.45) (2.91)
ALND: Axillary lymph node dissection; IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; C mixed: Mixed DuctalLobular Carcinoma; ER: oestrogenic receptors; PR: progesterone receptors; SLN: sentinel lymph node.
In the multivariate logistic regression analysis (OR (95% CI)), the presence of multifocality (2.16 (1.13e4.13), p ¼ 0.019) or lymphovascular infiltration (4.36 (2.43e7.82), p < 0.001), and the logTTL (1.22 (1.10e1.35), p < 0.001) were confirmed as independent predictors of more than 2 non-SLN MI with an overall fit (HosmereLemeshow test) of 0.359. Although tumour size also presented a trend as a predictor (coefficient of 0.018), it did not reach statistical signification (p ¼ 0.06). The ROC curve for this model, using TTL as main variable, is shown in Fig. 1, and their discriminating capacity, as
Table 2 Comparison of involved versus non-involved more than 2 non-SLN. Non-SLN negative/2 or less non-SLN positive (n ¼ 633) Age (years) Tumour size (mm) Multifocality Hystological type IDC NOS IDC specific ILC C mixed Grade I II III Lymphovascular infiltration ERþ PRþ HER2NEU þ Ki67 þ Immunophenotype Luminal A Luminal B Luminal B-Her Her-2-neu Triple negative Number of involved SLN Log TTL (copies/mL)
55.2 (12.2) 19.9 (11.7) 103 (16.3%)
More than 2 non-SLN positive (n ¼ 93) 56.2 (12.0) 26.7 (18.6) 28 (30.1%)
p-value
0.420a <0.001a 0.001b
544 26 55 8
(85.9%) (4.1%) (8.7%) (1.3%)
82 3 8 0
(88.1%) (3.2%) (8.6%) (0.0%)
0.560b
164 317 126 139
(25.9%) (50.1 %) (20.0%) (21.9%)
15 55 19 71
(16.1%) (59.1 %) (20.4%) (76.3%)
0.107b
<0.001b
481 403 55 271
(76.0%) (63.6%) (8.7%) (42.8%)
66 57 6 47
(71.0%) (61.3%) (6.4%) (50.5%)
0.700 0.578 0.579 0.018
216 219 29 15 24 1.1 9.66
(34.1%) (34.6%) (4.6%) (2.3%) (3.8%) (0.3) (2.69)
22 43 1 0 4 1.1 11.56
(23.7%) (46.2%) (1.1%) (0%) (4.3%) (0.4) (2.42)
0.030b
0.159a <0.001a
IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; C mixed: Mixed ductallobular carcinoma; ER: oestrogenic receptors; PR: progesterone receptors; SLN: sentinel lymph node; TTL: total tumoral load. a t-test. b Chi-square test.
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detect more than two affected nodes, as per the following equation:
Logit Pð > 2 nodesÞ ¼ 5:04 þ 1:38 LVI þ 0:709 multifocality þ 0:184 log TTL Fig. 2 shows sensitivity, specificity and predictive values as a function of the threshold defined on the TTL scale. This figure shows that both sensitivity and specificity exhibit a change in slope at about 10,000 copies/ml, and from that point on no relevant changes are seen in the NPV. There were 86 cases with TTL values between 5000 and 10,000 copies, which represent a 11.8% of the patients included in the study. Discussion
Fig. 1. ROC curve of the full three-variables model as predictors. Table 3 Discriminating capacity of the full multivariate model based on three variables and alternative reduced models depending on TTL. Model (more/less than 2 nSN)
AUC (95% CI)
p-valuea
Full model with three variables Log TTL excluded Log TTL alone
0.78 (0.72e0.83) 0.75 (0.69e0.80) 0.69 (0.64e0.75)
e 0.0001 0.0001
TTL: Tumour total load. SLN: Sentinel lymph nodes; CI: Confidence interval. a Difference with respect to the full model with three variables.
measured by the AUC, is shown in Table 3. Note that a predictability model including the correlation coefficients of the variables that exhibited statistical signification in the multivariate analysis allows us to estimate the probability to
We found statistically significant differences between patients with or without MI of more than 2 non-SLN in primary tumour size, multifocality, presence of lymphovascular infiltration, luminal B immunophenotype, positive proliferation index with ki67, and (log) TTL. Similar results were found in previous studies to predict any other non-SLN [30,31]. In our multivariate analysis, only multifocality, lymphovascular infiltration, and log TLL remained as independent predictors. This model was characterised by an AUC of 0.78 (0.725e0.835), which dropped to 0.75 (0.69e0.80) when log TTL was excluded as a predictor variable. The figure representing the model as a function of the threshold defined on the TTL scale shows that both sensitivity and specificity exhibit a change in slope at about 10,000 copies/ml, with no relevant changes in the NPV. Our results are in agreement with previous findings. A similar study with a lower sample size (n ¼ 185) showed, in multivariate analysis, that the mean SLN copy number, the number of macrometastatic SLN, and the presence of lymphovascular infiltration were informative for identifying non-SLN macrometastases [32]. In our study, with a higher sample size (n ¼ 726) focused on cases with more than two non-SLN detected after lymphadenectomy, the multivariate analysis did show an association with multifocality, TTL and lymphovascular infiltration. In the study by Ohi et al. [31], 130 patients with positive SLN that underwent complete ALND were investigated, and the CK19 mRNA copy number 5.0 103 in the SLN was the most significant predictor of non-SLN metastases
Fig. 2. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) for the presence of more than two affected nodes depending on the TLL.
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(p ¼ 0.003), while the CK19 mRNA copy number 1.0 105 in the SLN was the only independent predictor of 4 metastatic non-SLN nodes (p ¼ 0.014) [32]. Recently, a multivariate model to predict non-SLN MI based on log TTL, tumour size, number of affected SLN, presence of lymphovascular infiltration and Her2 status has been proposed [33,34]. We found that ki67 was a related variable, and we did not find Her2neu as a predictor, but all the remaining predictors in this study were also predictors in ours, and their model also had an AUC (0.718) very similar to ours (0.78). Finally, and concerning sensitivity, specificity and NPV values of our model as a function of the threshold defined on the TTL scale, our model showed similar results to those obtained when the analysis was repeated to predict the MI of two or more non-SLN (Giuliano et al. criteria [12]). The publication of the ACOSOG Z0011 study in 2011 [12] represented a great impact in breast cancer patients management, but it quickly raised some criticism due to methodological limitations [35]. The possibility of predicting axillary involvement based on OSNA measures may be important to surgeons that do not adopt what might be considered risky recommendations [12], as well as to guide radiotherapy [24e26]. Thus, the present work confirms that SLN assessment by OSNA allows the prediction of non-SLN MI, as it has been shown by other studies [31,32], suggesting that it would be feasible to avoid unnecessary lymphadenectomies when using systemic or radiotherapy regimens, and when we have to diagnose and take decisions, especially for radiotherapy, more based on the axillary state than on the main tumour. In addition, this study finds the possibility to predict more than two non-SLN knowing TTL in SN through OSNA technique following criteria of the ACOSOG Z0011 trial to avoid axillary lymphadenectomy. The present study also showed that choosing cut points in the TTL scale should be done for a specific definition of more than two non-SLN MI, by upgrading the 5000 copies cut point, classically associated with macrometastases, to a 10,000 copies cut point, with a parallel upgrading of specificity, and keeping the NPV of the procedure as part of the decision to use lymphadenectomy in cases with residual disease. If we accept that two metastatic lymph nodes may indicate ALND, applying TTL criteria with this new figure (10.000 copies) will increase in 11,8% to avoid ALND. In conclusion, molecular techniques such as OSNA procedure can improve breast cancer patients' management, because it allows discriminating between eligible and ineligible patients for lymphadenectomy or other axillary procedures. List of participants A) Qualify for authorship ~ ero-Madrona. Hospital Universitario “Virgen de la Antonio Pin Arrixaca”. Murcia. Spain. Guadalupe Ruiz-Merino. FFIS-IMIB. Murcia. Spain. tiva. Valencia. Spain. Laia Bernet. Hospital de Xa ~ a Miguel-Martínez. Hospital Universitario “General Begon Yagüe”. Burgos. Spain. Francisco Vicente-García. Complejo Hospitalario de Navarra. Pamplona. Spain. Amparo Viguri-Díaz. Hospital Txagorritxu. Vitoria. Spain. nez-Climent. Instituto Valenciano de Oncología. Julia Gime Valencia. Spain. B) Acquired data and contributed cases rica. Hospital Universitario de Albacete. Spain. Francisco Ande Jesús Acosta. Hospital “Santa Lucía”. Cartagena. Murcia. Spain. lez. Hospital “Puerta de Hierro”. Madrid. Spain. Carmen Gonza
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Irune Ruiz. Hospital de Donosti. San Sebasti an. Spain. s. Hospital Clínico de Valencia. Spain. Octavi Burgue n”. Palencia. Spain. Beatriz Torio. Hospital “Rio Carrio Rafael Cano. Hospital de Alzira. Valencia. Spain. ~ as. Complejo Hospitalario de Jae n. Spain. Basilio Duen Mª Angeles Torres-Nieto. Hospital “Rio Hortega”. Valladolid. Spain. David Hardisson. Hospital Universitario “La Paz”. Madrid. Spain. Jose Antonio Alberro. Onkologikoa. San Sebasti an. Spain. Luis Carrasco-Gonz alez. Hospital Universitario “JM Morales Meseguer”. Murcia. Spain. Sergi Vidal-Sicart. Hospital Clinic de Barcelona. Spain. Amalia Moreno. Hospital de Fuenlabrada. Madrid. Spain. Jesús Razquín. Hospital “Virgen de la Luz”. Cuenca. Spain. Pedro Marín-Rodriguez. Hospital Universitario “Reina Sofía”. Murcia. Spain. Angel Concha. Hospital Universitario “Virgen de las Nieves”. Granada. Spain. Ethical approval The study had been previously approved by the Ethics Committees of the participating centers. Conflict of interest statement None declared. References [1] Fentiman IS. Long-term follow-up of the first breast conservation trial: guy’ wide excision study. Breast 2000;9:5e8. [2] Lotze MT, Duncan MA, Gerber LH, Woltering EA, Rosenberg SA. Early versus delayed shoulder motion following axillary dissection: a randomized prospective study. Ann Surg 1981;193:288e95. [3] Ivens D, Hoe AL, Podd TJ, Hamilton CR, Taylor I, Royle GT. Assessment of morbidity from complete axillary dissection. Br J Cancer 1992;66:136e8. [4] Lucci A, McCall LM, Beitsch PD, Whitworth PW, Reintgen DS, Blumencranz PW, et al. Surgical complications associated with sentinel lymph node dissection (SLND) plus axillary lymph node dissection compared with SLND alone in the American college of surgeons oncology group trial Z0011. J Clin Oncol 2007;25:3657e63. [5] Lyman GH, Giuliano AE, Somerfield MR, Benson 3rd AB, Bodurka DC, Burstein HJ, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol 2005;23:7703e20. [6] Viale G, Maiorano E, Mazzarol G, Zurrida S, Galimberti V, Luini A, et al. Histologic detection and clinical implications of micrometastases in axillary sentinel lymph nodes for patients with breast carcinoma. Cancer 2001;92: 1378e84. [7] Den Bakker MA, van Weeszenberg A, de Kanter AY, Beverdam FH, Pritchard C, van der Kwast TH, et al. Non-sentinel lymph node involvement in patients with breast cancer and sentinel node micrometastasis; too early to abandon axillary clearance. J Clin Pathol 2002;55:932e5. [8] Langer I, Guller U, Viehl CT, Moch H, Wight E, Harder F, et al. Axillary lymph node dissection for sentinel lymph node micrometastases may be safely omitted in early-stage breast cancer patients: long-term outcomes of a prospective study. Ann Surg Oncol 2009;16:59e67. [9] Hansen NM, Grube B, Ye X, Turner RR, Brenner RJ, Sim M-S, et al. Impact of micrometastases in the sentinel node of patients with invasive breast cancer. J Clin Oncol 2009;27:4679e84. [10] Yegiyants S, Romero LM, Haigh PI, DiFronzo LA. Completion axillary lymph node dissection not required for regional control in patients with breast cancer who have micrometastases in a sentinel node. Arch Surg 2010;145: 564e9. [11] De Boer M, van Deurzen CHM, van Dijck JAAM, Borm GF, van Diest PJ, Adang EMM, et al. Micrometastases or isolated tumor cells and the outcome of breast cancer. N Engl J Med 2009;361:653e63. [12] Giuliano AE, Hunt KK, Ballman KV, Beitsch PD, Whitworth PW, Blumencranz PW, et al. Axillary dissection vs no axillary dissection in women with invasive breast cancer and sentinel node metastasis: a randomized clinical trial. JAMA 2011;305:569e75. ~ as B, Matias-Guiu X, Morell L, et al. [13] Bernet L, Cano R, Martinez M, Duen Diagnosis of the sentinel lymph node in breast cancer: a reproducible molecular method: a multicentric Spanish study. Histopathology 2011;58:863e9.
864
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[14] Tamaki Y, Akiyama F, Iwase T, Kaneko T, Tsuda H, Sato K, et al. Molecular detection of lymph node metastases in breast cancer patients: results of a multicenter trial using the one-step nucleic acid amplification assay. Clin Cancer Res 2009;15:2879e84. [15] Cserni G. Intraoperative analysis of sentinel lymph nodes in breast cancer by one-step nucleic acid amplification. J Clin Pathol 2012;65:193e9. n-Paredes MP, Carrasco-Gonza lez L, Cha ves-Benito A, Campillo-Soto A, [16] Guille Carrillo A, Aguayo-Albasini JL. One-step nucleic acid amplification (OSNA) assay for sentinel lymph node metastases as an alternative to conventional postoperative histology in breast cancer: a cost-benefit analysis. Cir Esp 2011;89:456e62. [17] Ganaraj A, Kuhn JA, Jones RC, Grant MD, Andrews VR, Knox SM, et al. Predictors for nonsentinel node involvement in breast cancer patients with micrometastases in the sentinel lymph node. Proc 2003;16:3e6. [18] Fournier K, Schiller A, Perry RR, Laronga C. Micrometastasis in the sentinel lymph node of breast cancer does not mandate completion axillary dissection. Ann Surg 2004;239:859e63. discussion 863e5. [19] Rutledge H, Davis J, Chiu R, Cibull M, Brill Y, McGrath P, et al. Sentinel node micrometastasis in breast carcinoma may not be an indication for complete axillary dissection. Mod Pathol 2005;18:762e8. [20] Kumar S, Bramlage M, Jacks LM, Goldberg JI, Patil SM, Giri DD, et al. Minimal disease in the sentinel lymph node: how to best measure sentinel node micrometastases to predict risk of additional non-sentinel lymph node disease. Ann Surg Oncol 2010;17:2909e19. [21] Straver ME, Meijnen P, van Tienhoven G, van de Velde CJ, Mansel RE, Bogaerts J, et al. Sentinel node identification rate and nodal involvement in the EORTC 10981-22023 AMAROS trial. Ann Surg Oncol 2010;17:1854e61. [22] Wasif N, Maggard MA, Ko CY, Giuliano AE. Underuse of axillary dissection for the management of sentinel node micrometastases in breast cancer. Arch Surg 2010;145:161e6. [23] Reed J, Rosman M, Verbanac KM, Mannie A, Cheng Z, Tafra L. Prognostic implications of isolated tumor cells and micrometastases in sentinel nodes of patients with invasive breast cancer: 10-year analysis of patients enrolled in the prospective East Carolina University/Anne Arundel Medical Center Sentinel Node Mult. J Am Coll Surg 2009;208:333e40. LB, Rojo F, Hardisson D, Iturriagagoitia AC, Panade s MJ, Velasco A, et al. [24] Vegue Comparison of molecular analysis and histopathology for axillary lymph node staging in primary breast cancer: results of the B-CLOSER-I study. Diagn Mol Pathol 2012;21:69e76.
[25] Caudle AS, Hunt KK, Kuerer HM, Meric-Bernstam F, Lucci A, Bedrosian I, et al. Multidisciplinary considerations in the implementation of the findings from the American College of Surgeons Oncology Group (ACOSOG) Z0011 study: a practice-changing trial. Ann Surg Oncol 2011;18:2407e12. [26] Caudle AS, Hunt KK, Tucker SL, Hoffman K, Gainer SM, Lucci A, et al. American College of Surgeons Oncology Group (ACOSOG) Z0011: impact on surgeon practice patterns. Ann Surg Oncol 2012;19:3144e51. [27] Haffty BG, Hunt KK, Harris JR, Buchholz TA. Positive sentinel nodes without axillary dissection: implications for the radiation oncologist. J Clin Oncol 2011;29:4479e81. ~ ero A, Gime nez J, Merck B, V [28] Pin azquez C. Consensus meeting on selective biopsy of the sentinel node in breast cancer. Spanish Society of Senology and Breast Disease. Rev Esp Med Nucl 2007;26:176e80. [29] Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama F, et al. One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Cancer Res 2007;13: 4807e16. [30] Bloom H, Richardson WW. Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer 1957;11:359e77. [31] Ohi Y, Umekita Y, Sagara Y, Rai Y, Yotsumoto D, Matsukata A, et al. Whole sentinel lymph node analysis by a molecular assay predicts axillary node status in breast cancer. Br J Cancer 2012;107:1239e43. [32] Osako T, Iwase T, Kimura K, Horii R, Akiyama F. Sentinel node tumor burden quantified based on cytokeratin 19 mRNA copy number predicts non-sentinel node metastases in breast cancer: molecular whole-node analysis of all removed nodes. Eur J Cancer 2013;49:1187e95. rez-Hoyos S, Ramos M, Sancho M, Xercavins J, [33] Espinosa-Bravo M, Sansano I, Pe et al. Prediction of non-sentinel lymph node metastasis in early breast cancer by assessing total tumoral load in the sentinel lymph node by molecular assay. Eur J Surg Oncol 2013;39:766e73. [34] Peg V, Espinosa-Bravo M, Vieites B, Vilardell F, Antúnez JR, de Salas MS, et al. Intraoperative molecular analysis of total tumor load in sentinel lymph node: a new predictor of axillary status in early breast cancer patients. Breast Cancer Res Treat 2013;139:87e93. [35] Latosinsky S, Berrang TS, Cutter CS, George R, Olivotto I, Julian TB, et al. CAGS and ACS evidence based reviews in surgery. 40. Axillary dissection versus no axillary dissection in women with invasive breast cancer and sentinel node metastasis. Can J Surg 2012;55:66e9.
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Original article
Tumoral load quantification of positive sentinel lymph nodes in breast cancer to predict more than two involved nodes ~ ero-Madrona a, *, Guadalupe Ruiz-Merino b, Laia Bernet c, Antonio Pin ~ a Miguel-Martínez d, Francisco Vicente-García e, María A. Viguri-Díaz f, Begon nez-Climent g Julia Gime a
Department of Surgery, “Virgen de la Arrixaca” University Hospital, 30120 Murcia, Spain n, 9, 1ª Planta, 30120 Murcia, Spain Department of Statistics, FFIS-IMIB, Luis Fontes Paga tiva, Crtra de Xa tiva a Silla, Km 1, Valencia, Spain Department of Pathology, Hospital of Xa d Department of Nuclear Medicine, University Hospital, Hijos de Santiago Rodriguez, 16, 09002 Burgos, Spain e Department of Surgery, Complejo Hospitalario de Navarra, La Arboleda, 4, Cizur Menor, 31190 Pamplona, Spain f Department of Pathology, University Hospital Txagorritxu, Jose Atxotegui s/n, 01009 Vitoria, Spain g n Ba guena, 8, 46009 Valencia, Spain n Instituto Valenciano de Oncología, Prof. Beltra Department of Surgery, Fundacio b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 April 2014 Received in revised form 9 September 2014 Accepted 12 September 2014 Available online 3 October 2014
Aim: One-Step Nucleic Acid Amplification (OSNA) can detect isolated tumour loads in axillary lymph nodes of breast cancer patients. We investigated the predictability of the non-sentinel lymph node (SLN) metastatic involvement (MI) based on the OSNA SLN assessment in surgical invasive breast cancer. Methods: We studied surgical breast invasive carcinoma patients, not taking neoadjuvant chemotherapy, having SLN positive by OSNA and having received axillary lymphadenectomy. Age, basic histopathological, immunohistochemical, SLN biopsy and lymphadenectomy data were compared between patients with or without MI of more than 2 non-SLN in both univariate and multivariate analyses. The discriminating capacity of the multivariate model was characterized by the ROC AUC. Results: 726 patients from 23 centers in Spain aged 55.3 ± 12.2 years were analysed. The univariate analysis comparing patients with or without MI of more than 2 non-SLN detected statistically significant differences in primary tumour size, multifocality, presence of lymphovascular infiltration, positive proliferation index with ki67, immunophenotype and logTTL (Tumour Total Load). The multivariate logistic analyses (OR (95% CI)) confirmed multifocality (2.16 (1.13e4.13), p ¼ 0.019), lymphovascular infiltration (4.36 (2.43e7.82), p < 0.001) and logTTL (1.22 (1.10e1.35), p < 0.001) as independent predictors, and exhibit an AUC (95% CI) of 0.78 (0.72e0.83) with an overall fit (HosmereLemeshow test) of 0.359. A change in the slope of both sensitivity and specificity is observed at about 10,000 copies/mL, without relevant changes in the Negative Predictive Values. Conclusions: Using OSNA technique, the MI of more than 2 non-SLN can be reliably predicted. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Breast neoplasms Intraoperative procedures Keratin 19 mRNA copy number One-step nucleic acid amplification assay Sentinel lymph-node biopsy
Introduction In the last twenty years, the election of the axillary locoregional treatment for breast cancer has become a key decision that can determine global and disease-free survival [1]. Studies initiated in
* Corresponding author. Tel.: þ34 968369677; fax: þ34 968395537. E-mail addresses: [email protected], [email protected] ~ ero-Madrona), [email protected] (G. Ruiz-Merino), [email protected] (A. Pin (L. Bernet), [email protected] (B. Miguel-Martínez), [email protected] (F. VicenteGarcía), [email protected] (M.A. Viguri-Díaz), julia. nez-Climent). [email protected] (J. Gime http://dx.doi.org/10.1016/j.breast.2014.09.005 0960-9776/© 2014 Elsevier Ltd. All rights reserved.
the 1980s suggested that the Axillary Lymph Node Dissection (ALND) entails an unacceptable risk of serious complications [2,3]. With the objective of benefit maximization and risk minimization, the standard ALND was reconsidered, and the Sentinel Lymph Node Dissection (SLND) technique was developed as a more precise technique in the staging of tumour draining axillary nodes, with less morbidity than ALND [4]. Nowadays, the SLND is generally accepted as the sole management for patients whose sentinel lymph nodes (SLNs) are unaffected according to histology or OSNA (One-Step Nucleic Acid Amplification), while ALND remains the standard of care for patients whose SLNs contain metastases [5]. In any case, choice of treatment implies other problems, and at
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present there is a need for further treatment optimization and standardization, so as to avoid unnecessary risks for patients. The intraoperative selective biopsy of SLN has represented a significant increase in the detection of micrometastases and submicrometastases as compared with previous periods [6,7]. Recent studies, both retrospective [8] and prospective [9,10], suggest the possibility of not carrying out ALND when only micrometastases are found in the SLN, especially in certain subgroups of good prognosis [11], or even in the case of macroscopic limited nodal disease [12]. More in detail, the results of the ACOSOG Z0011 study suggested that it could be possible to prevent ALND, even when the macroscopic nodal disease affected up to two SLN [12]. Thus, the interest would be to characterise which cases would be less probably affected by axillary disease beyond the two SLN, and more specifically in the case of a positive selective biopsy of SLN with an adequate negative predictive value (NPV), useful to predict their probability of appearance. In this context, advances in diagnostic technology based on molecular methods for the analysis of SLN might be essential to improve therapeutic management. OSNA is not able to detect isolate tumoral cells but only micro and macrometastases load through detection of mRNA CK19 expression in the node [13,14]. The OSNA method has shown a 96% concordance rate with detailed histopathology complemented with immunohistochemistry, a low false-negative rate, and a very low false-positive rate, what makes this technique suitable for the intraoperative evaluation of SLN [15]. On the other hand, intraoperative molecular analysis for SLN metastases using the OSNA method shortens admission time and the duration of surgery, and saves 439.67 V per patient in Spain [16]. It is tempting to change the gold standard for comparisons between methods, and when this is done, histology seems to come out as a weaker test for the identification of metastases. In any case, despite the advantage of detecting even very limited nodal involvement, the implications of this high sensitivity in deciding the convenience of ALND, or its magnitude, remain unclear. While some studies have suggested not to perform ALND in case of a low metastatic involvement (MI) rate of non-SLN [17e19], others have pointed out that this may occur in more than 20% of cases, and therefore they do not recommend to avoid lymphadenectomy [20e24]. Nevertheless, the sensitivity of the OSNA method clearly represents an opportunity for a fine assessment of the tumour load in axillary nodes, to determine a threshold for avoiding lymphadenectomy, and also to guide radiotherapy so as to reduce its associated morbidities [25e27]. The aim of the present work was to characterize the relationship between the tumour load in positive/s SLN, using OSNA method, and the presence of disease in two or more additional non-SLN in breast cancer patients, so as to establish a threshold to optimize the prediction of non-SLN MI in order to avoid ALND.
Methods Study design We conducted a multicenter observational study in patients with invasive breast carcinoma subjected to a selective SLN biopsy.
Setting The study was conducted in Breast Surgery Units from 23 Spanish hospitals. The study had been previously approved by the Ethics Committees of the participating centers. Medical records provided the necessary data for this study.
Participants We included all patients with a positive SLN biopsy according to the OSNA technique, that later underwent an ALND of at least 10 lymph nodes. We excluded patients with negative SLN, those that had received neoadjuvant chemotherapy, or that had SLN assessed with a method different from OSNA, or when the histopathological assessment of isolated lymph nodes from the ALND was other than usual (halved in two slices). SLN and ALND evaluation The Sentinel Lymph Node Biopsy was performed in the participating centres following the recommendations of the consensus document from the Senology and Mammary Pathology Spanish Society [28]. The OSNA assay was performed for the isolated sentinel lymph nodes analysis. The OSNA assay for lymph nodes has been previously described in detail [29]. Briefly, the number of mRNACK19 copies per ml in the measurement sample was calculated; these copy numbers were used as basis to determine the result. The cutoffs for negative/positive and micro-/macrometastases were set at 250 and 5000 copies/ml, respectively. The axillary lymph nodes from the lymphadenectomy were studied following conventional protocol, that implies isolation of all the nodes, and each one was processed by bipartition and histopathological study of one 4 m section of every piece from both sections. Immunohistochemistry for cytokeratine (AE1/AE3) was used only in doubtful cases. Variables and definitions For each patient included in the study, we recorded the number of biopsied SLNs (as selected by the surgeon intraoperatively), the total tumour load (TTL) from SLNs (mRNA CK19 copies/mL), the number of nodes isolated in ALND, and the number of those that were affected as per histopathological assessment. Cases were pooled for analysis in those patients with more than two affected nodes (following the lymphadenectomy criteria in recent series [12]). Other variables recorded were: patient's age, primary tumour size, histological type, histological grade (according to ScarffBloom-Richardson [30]), lymphovascular infiltration, bilaterality and immunophenotype classification (expression of oestrogenic and progesterone receptors, considering more than 10% dyed cells as positive, overexpression of Her-2-neu, considering þþþ and þþ with positive FISH as positive, and the ki67, considering>14% dyed cells as positive). Luminal A immunophenotype was described as ER and PR expression with low ki67 figures; Luminal B, when there was ER and PR expression with ki67 higher than 14%; Luminal BeH, when Her2neu overexpression was added to Luminal B profile; Her2neu was defined as Her2neu overexpression without ER or PR expression; and triple negative profile, when there was neither ER or PR expression or Her2. Statistical methods The sample size was determined by the availability of eligible cases in the participating centers during the study period, and no formal sample size was estimated. As a first step, a univariate analysis was conducted to compare study variables in patients with or without MI of more than 2 nonSLN, using the Student t-test, the Pearson chi-square test, or the Fisher's exact test as suitable. Then, a multivariate logistic
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regression model was built with those variables that showed significant results in the previous univariate analysis. A stepwise selection procedure was used to select the model predictors, with p < 0.05 in Wald tests as entry and retain criteria. We characterised the discriminating ability of the resulting model, and of some alternative reduced models, by plotting the corresponding ROC curves and computing the area under the curves (AUC). The data are described by means and standard deviations or counts and percentages, as suitable. Results from statistical tests were considered significant if two-sided p < 0.05. The statistical analysis was carried out with SPSS v.18.0.
SLN nodes were isolated in ALND (n ¼ 34), if they received neoadjuvant chemotherapy (n ¼ 16), had SLN assessed with a method different from OSNA, or had non-conventional histopathologic assessment of non-SLN. The total number of patients included in the analysis was 726.
Results
Univariate analysis
Participants
The univariate analysis comparing patients with or without MI of more than 2 non-SLN detected statistically significant differences in the primary tumour size, multifocality, presence of lymphovascular infiltration, positive proliferation index with ki67, luminal B immunophenotype, and (log) TTL (see Table 2).
From 2000 to 2012, 797 patients from 23 centers with a diagnosis of invasive breast carcinoma were operated, and sentinel lymph nodes biopsied. Patients were excluded if less than 10 non-
Descriptive data The age of patients was 55.3 ± 12.2 years (range: 28e89), and the size of the primary tumour was of 20.8 ± 13.0 mm (range: 1e150). Patients' characteristics are shown in Table 1.
Multivariate analysis Table 1 Patient's characteristics. Data are expressed as mean (SD) or n (%) as appropriate. Age (years) Hystological type IDC ILC C mixed Size (mm) Focality Unifocal Multifocal Not available Bilaterality Differentiation Good Moderate Scarce Not available Lymphovascular infiltration Yes No Not available ER Positive Negative Not available PR Positive Negative Not available HER2NEU Positive Negative Not available Ki67 Positive Negative Not available Immunophenotype Luminal A Luminal B Luminal BH HER Triple negative Not available Number of isolated SLN Number of affected SLN Number of lymphadenectomy isolated nodes Involved nodes in ALND
55.28 (12.20) 655 63 8 20.85
(90.2%) (8.7%) (1.1%) (13.01)
592 131 3 8
(81.5%) (18.1%) (0.4%) (1.1%)
179 372 145 30
(24.6%) (51.3%) (20.0%) (4.1%)
211 (29.0%) 365 (50.3%) 150 (20.7%) 547 (75.3%) 49 (6.8%) 130 (17.9%) 460 (63.3%) 132 (18.2%) 134 (18.5%) 61 (8.4%) 517 (71.2%) 148 (20.3%) 318 (43.8%) 262 (36.1%) 146 (20.1%) 238 262 30 15 33 148 1.73 1.09 17.30 1.29
(32.8%) (36.1%) (4.1%) (2.0%) (4.5%) (20.5%) (0.846) (0.293) (5.45) (2.91)
ALND: Axillary lymph node dissection; IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; C mixed: Mixed DuctalLobular Carcinoma; ER: oestrogenic receptors; PR: progesterone receptors; SLN: sentinel lymph node.
In the multivariate logistic regression analysis (OR (95% CI)), the presence of multifocality (2.16 (1.13e4.13), p ¼ 0.019) or lymphovascular infiltration (4.36 (2.43e7.82), p < 0.001), and the logTTL (1.22 (1.10e1.35), p < 0.001) were confirmed as independent predictors of more than 2 non-SLN MI with an overall fit (HosmereLemeshow test) of 0.359. Although tumour size also presented a trend as a predictor (coefficient of 0.018), it did not reach statistical signification (p ¼ 0.06). The ROC curve for this model, using TTL as main variable, is shown in Fig. 1, and their discriminating capacity, as
Table 2 Comparison of involved versus non-involved more than 2 non-SLN. Non-SLN negative/2 or less non-SLN positive (n ¼ 633) Age (years) Tumour size (mm) Multifocality Hystological type IDC NOS IDC specific ILC C mixed Grade I II III Lymphovascular infiltration ERþ PRþ HER2NEU þ Ki67 þ Immunophenotype Luminal A Luminal B Luminal B-Her Her-2-neu Triple negative Number of involved SLN Log TTL (copies/mL)
55.2 (12.2) 19.9 (11.7) 103 (16.3%)
More than 2 non-SLN positive (n ¼ 93) 56.2 (12.0) 26.7 (18.6) 28 (30.1%)
p-value
0.420a <0.001a 0.001b
544 26 55 8
(85.9%) (4.1%) (8.7%) (1.3%)
82 3 8 0
(88.1%) (3.2%) (8.6%) (0.0%)
0.560b
164 317 126 139
(25.9%) (50.1 %) (20.0%) (21.9%)
15 55 19 71
(16.1%) (59.1 %) (20.4%) (76.3%)
0.107b
<0.001b
481 403 55 271
(76.0%) (63.6%) (8.7%) (42.8%)
66 57 6 47
(71.0%) (61.3%) (6.4%) (50.5%)
0.700 0.578 0.579 0.018
216 219 29 15 24 1.1 9.66
(34.1%) (34.6%) (4.6%) (2.3%) (3.8%) (0.3) (2.69)
22 43 1 0 4 1.1 11.56
(23.7%) (46.2%) (1.1%) (0%) (4.3%) (0.4) (2.42)
0.030b
0.159a <0.001a
IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; C mixed: Mixed ductallobular carcinoma; ER: oestrogenic receptors; PR: progesterone receptors; SLN: sentinel lymph node; TTL: total tumoral load. a t-test. b Chi-square test.
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detect more than two affected nodes, as per the following equation:
Logit Pð > 2 nodesÞ ¼ 5:04 þ 1:38 LVI þ 0:709 multifocality þ 0:184 log TTL Fig. 2 shows sensitivity, specificity and predictive values as a function of the threshold defined on the TTL scale. This figure shows that both sensitivity and specificity exhibit a change in slope at about 10,000 copies/ml, and from that point on no relevant changes are seen in the NPV. There were 86 cases with TTL values between 5000 and 10,000 copies, which represent a 11.8% of the patients included in the study. Discussion
Fig. 1. ROC curve of the full three-variables model as predictors. Table 3 Discriminating capacity of the full multivariate model based on three variables and alternative reduced models depending on TTL. Model (more/less than 2 nSN)
AUC (95% CI)
p-valuea
Full model with three variables Log TTL excluded Log TTL alone
0.78 (0.72e0.83) 0.75 (0.69e0.80) 0.69 (0.64e0.75)
e 0.0001 0.0001
TTL: Tumour total load. SLN: Sentinel lymph nodes; CI: Confidence interval. a Difference with respect to the full model with three variables.
measured by the AUC, is shown in Table 3. Note that a predictability model including the correlation coefficients of the variables that exhibited statistical signification in the multivariate analysis allows us to estimate the probability to
We found statistically significant differences between patients with or without MI of more than 2 non-SLN in primary tumour size, multifocality, presence of lymphovascular infiltration, luminal B immunophenotype, positive proliferation index with ki67, and (log) TTL. Similar results were found in previous studies to predict any other non-SLN [30,31]. In our multivariate analysis, only multifocality, lymphovascular infiltration, and log TLL remained as independent predictors. This model was characterised by an AUC of 0.78 (0.725e0.835), which dropped to 0.75 (0.69e0.80) when log TTL was excluded as a predictor variable. The figure representing the model as a function of the threshold defined on the TTL scale shows that both sensitivity and specificity exhibit a change in slope at about 10,000 copies/ml, with no relevant changes in the NPV. Our results are in agreement with previous findings. A similar study with a lower sample size (n ¼ 185) showed, in multivariate analysis, that the mean SLN copy number, the number of macrometastatic SLN, and the presence of lymphovascular infiltration were informative for identifying non-SLN macrometastases [32]. In our study, with a higher sample size (n ¼ 726) focused on cases with more than two non-SLN detected after lymphadenectomy, the multivariate analysis did show an association with multifocality, TTL and lymphovascular infiltration. In the study by Ohi et al. [31], 130 patients with positive SLN that underwent complete ALND were investigated, and the CK19 mRNA copy number 5.0 103 in the SLN was the most significant predictor of non-SLN metastases
Fig. 2. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) for the presence of more than two affected nodes depending on the TLL.
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(p ¼ 0.003), while the CK19 mRNA copy number 1.0 105 in the SLN was the only independent predictor of 4 metastatic non-SLN nodes (p ¼ 0.014) [32]. Recently, a multivariate model to predict non-SLN MI based on log TTL, tumour size, number of affected SLN, presence of lymphovascular infiltration and Her2 status has been proposed [33,34]. We found that ki67 was a related variable, and we did not find Her2neu as a predictor, but all the remaining predictors in this study were also predictors in ours, and their model also had an AUC (0.718) very similar to ours (0.78). Finally, and concerning sensitivity, specificity and NPV values of our model as a function of the threshold defined on the TTL scale, our model showed similar results to those obtained when the analysis was repeated to predict the MI of two or more non-SLN (Giuliano et al. criteria [12]). The publication of the ACOSOG Z0011 study in 2011 [12] represented a great impact in breast cancer patients management, but it quickly raised some criticism due to methodological limitations [35]. The possibility of predicting axillary involvement based on OSNA measures may be important to surgeons that do not adopt what might be considered risky recommendations [12], as well as to guide radiotherapy [24e26]. Thus, the present work confirms that SLN assessment by OSNA allows the prediction of non-SLN MI, as it has been shown by other studies [31,32], suggesting that it would be feasible to avoid unnecessary lymphadenectomies when using systemic or radiotherapy regimens, and when we have to diagnose and take decisions, especially for radiotherapy, more based on the axillary state than on the main tumour. In addition, this study finds the possibility to predict more than two non-SLN knowing TTL in SN through OSNA technique following criteria of the ACOSOG Z0011 trial to avoid axillary lymphadenectomy. The present study also showed that choosing cut points in the TTL scale should be done for a specific definition of more than two non-SLN MI, by upgrading the 5000 copies cut point, classically associated with macrometastases, to a 10,000 copies cut point, with a parallel upgrading of specificity, and keeping the NPV of the procedure as part of the decision to use lymphadenectomy in cases with residual disease. If we accept that two metastatic lymph nodes may indicate ALND, applying TTL criteria with this new figure (10.000 copies) will increase in 11,8% to avoid ALND. In conclusion, molecular techniques such as OSNA procedure can improve breast cancer patients' management, because it allows discriminating between eligible and ineligible patients for lymphadenectomy or other axillary procedures. List of participants A) Qualify for authorship ~ ero-Madrona. Hospital Universitario “Virgen de la Antonio Pin Arrixaca”. Murcia. Spain. Guadalupe Ruiz-Merino. FFIS-IMIB. Murcia. Spain. tiva. Valencia. Spain. Laia Bernet. Hospital de Xa ~ a Miguel-Martínez. Hospital Universitario “General Begon Yagüe”. Burgos. Spain. Francisco Vicente-García. Complejo Hospitalario de Navarra. Pamplona. Spain. Amparo Viguri-Díaz. Hospital Txagorritxu. Vitoria. Spain. nez-Climent. Instituto Valenciano de Oncología. Julia Gime Valencia. Spain. B) Acquired data and contributed cases rica. Hospital Universitario de Albacete. Spain. Francisco Ande Jesús Acosta. Hospital “Santa Lucía”. Cartagena. Murcia. Spain. lez. Hospital “Puerta de Hierro”. Madrid. Spain. Carmen Gonza
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Irune Ruiz. Hospital de Donosti. San Sebasti an. Spain. s. Hospital Clínico de Valencia. Spain. Octavi Burgue n”. Palencia. Spain. Beatriz Torio. Hospital “Rio Carrio Rafael Cano. Hospital de Alzira. Valencia. Spain. ~ as. Complejo Hospitalario de Jae n. Spain. Basilio Duen Mª Angeles Torres-Nieto. Hospital “Rio Hortega”. Valladolid. Spain. David Hardisson. Hospital Universitario “La Paz”. Madrid. Spain. Jose Antonio Alberro. Onkologikoa. San Sebasti an. Spain. Luis Carrasco-Gonz alez. Hospital Universitario “JM Morales Meseguer”. Murcia. Spain. Sergi Vidal-Sicart. Hospital Clinic de Barcelona. Spain. Amalia Moreno. Hospital de Fuenlabrada. Madrid. Spain. Jesús Razquín. Hospital “Virgen de la Luz”. Cuenca. Spain. Pedro Marín-Rodriguez. Hospital Universitario “Reina Sofía”. Murcia. Spain. Angel Concha. Hospital Universitario “Virgen de las Nieves”. Granada. Spain. Ethical approval The study had been previously approved by the Ethics Committees of the participating centers. Conflict of interest statement None declared. References [1] Fentiman IS. Long-term follow-up of the first breast conservation trial: guy’ wide excision study. Breast 2000;9:5e8. [2] Lotze MT, Duncan MA, Gerber LH, Woltering EA, Rosenberg SA. Early versus delayed shoulder motion following axillary dissection: a randomized prospective study. Ann Surg 1981;193:288e95. [3] Ivens D, Hoe AL, Podd TJ, Hamilton CR, Taylor I, Royle GT. Assessment of morbidity from complete axillary dissection. Br J Cancer 1992;66:136e8. [4] Lucci A, McCall LM, Beitsch PD, Whitworth PW, Reintgen DS, Blumencranz PW, et al. Surgical complications associated with sentinel lymph node dissection (SLND) plus axillary lymph node dissection compared with SLND alone in the American college of surgeons oncology group trial Z0011. J Clin Oncol 2007;25:3657e63. [5] Lyman GH, Giuliano AE, Somerfield MR, Benson 3rd AB, Bodurka DC, Burstein HJ, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol 2005;23:7703e20. [6] Viale G, Maiorano E, Mazzarol G, Zurrida S, Galimberti V, Luini A, et al. Histologic detection and clinical implications of micrometastases in axillary sentinel lymph nodes for patients with breast carcinoma. Cancer 2001;92: 1378e84. [7] Den Bakker MA, van Weeszenberg A, de Kanter AY, Beverdam FH, Pritchard C, van der Kwast TH, et al. Non-sentinel lymph node involvement in patients with breast cancer and sentinel node micrometastasis; too early to abandon axillary clearance. J Clin Pathol 2002;55:932e5. [8] Langer I, Guller U, Viehl CT, Moch H, Wight E, Harder F, et al. Axillary lymph node dissection for sentinel lymph node micrometastases may be safely omitted in early-stage breast cancer patients: long-term outcomes of a prospective study. Ann Surg Oncol 2009;16:59e67. [9] Hansen NM, Grube B, Ye X, Turner RR, Brenner RJ, Sim M-S, et al. Impact of micrometastases in the sentinel node of patients with invasive breast cancer. J Clin Oncol 2009;27:4679e84. [10] Yegiyants S, Romero LM, Haigh PI, DiFronzo LA. Completion axillary lymph node dissection not required for regional control in patients with breast cancer who have micrometastases in a sentinel node. Arch Surg 2010;145: 564e9. [11] De Boer M, van Deurzen CHM, van Dijck JAAM, Borm GF, van Diest PJ, Adang EMM, et al. Micrometastases or isolated tumor cells and the outcome of breast cancer. N Engl J Med 2009;361:653e63. [12] Giuliano AE, Hunt KK, Ballman KV, Beitsch PD, Whitworth PW, Blumencranz PW, et al. Axillary dissection vs no axillary dissection in women with invasive breast cancer and sentinel node metastasis: a randomized clinical trial. JAMA 2011;305:569e75. ~ as B, Matias-Guiu X, Morell L, et al. [13] Bernet L, Cano R, Martinez M, Duen Diagnosis of the sentinel lymph node in breast cancer: a reproducible molecular method: a multicentric Spanish study. Histopathology 2011;58:863e9.
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[14] Tamaki Y, Akiyama F, Iwase T, Kaneko T, Tsuda H, Sato K, et al. Molecular detection of lymph node metastases in breast cancer patients: results of a multicenter trial using the one-step nucleic acid amplification assay. Clin Cancer Res 2009;15:2879e84. [15] Cserni G. Intraoperative analysis of sentinel lymph nodes in breast cancer by one-step nucleic acid amplification. J Clin Pathol 2012;65:193e9. n-Paredes MP, Carrasco-Gonza lez L, Cha ves-Benito A, Campillo-Soto A, [16] Guille Carrillo A, Aguayo-Albasini JL. One-step nucleic acid amplification (OSNA) assay for sentinel lymph node metastases as an alternative to conventional postoperative histology in breast cancer: a cost-benefit analysis. Cir Esp 2011;89:456e62. [17] Ganaraj A, Kuhn JA, Jones RC, Grant MD, Andrews VR, Knox SM, et al. Predictors for nonsentinel node involvement in breast cancer patients with micrometastases in the sentinel lymph node. Proc 2003;16:3e6. [18] Fournier K, Schiller A, Perry RR, Laronga C. Micrometastasis in the sentinel lymph node of breast cancer does not mandate completion axillary dissection. Ann Surg 2004;239:859e63. discussion 863e5. [19] Rutledge H, Davis J, Chiu R, Cibull M, Brill Y, McGrath P, et al. Sentinel node micrometastasis in breast carcinoma may not be an indication for complete axillary dissection. Mod Pathol 2005;18:762e8. [20] Kumar S, Bramlage M, Jacks LM, Goldberg JI, Patil SM, Giri DD, et al. Minimal disease in the sentinel lymph node: how to best measure sentinel node micrometastases to predict risk of additional non-sentinel lymph node disease. Ann Surg Oncol 2010;17:2909e19. [21] Straver ME, Meijnen P, van Tienhoven G, van de Velde CJ, Mansel RE, Bogaerts J, et al. Sentinel node identification rate and nodal involvement in the EORTC 10981-22023 AMAROS trial. Ann Surg Oncol 2010;17:1854e61. [22] Wasif N, Maggard MA, Ko CY, Giuliano AE. Underuse of axillary dissection for the management of sentinel node micrometastases in breast cancer. Arch Surg 2010;145:161e6. [23] Reed J, Rosman M, Verbanac KM, Mannie A, Cheng Z, Tafra L. Prognostic implications of isolated tumor cells and micrometastases in sentinel nodes of patients with invasive breast cancer: 10-year analysis of patients enrolled in the prospective East Carolina University/Anne Arundel Medical Center Sentinel Node Mult. J Am Coll Surg 2009;208:333e40. LB, Rojo F, Hardisson D, Iturriagagoitia AC, Panade s MJ, Velasco A, et al. [24] Vegue Comparison of molecular analysis and histopathology for axillary lymph node staging in primary breast cancer: results of the B-CLOSER-I study. Diagn Mol Pathol 2012;21:69e76.
[25] Caudle AS, Hunt KK, Kuerer HM, Meric-Bernstam F, Lucci A, Bedrosian I, et al. Multidisciplinary considerations in the implementation of the findings from the American College of Surgeons Oncology Group (ACOSOG) Z0011 study: a practice-changing trial. Ann Surg Oncol 2011;18:2407e12. [26] Caudle AS, Hunt KK, Tucker SL, Hoffman K, Gainer SM, Lucci A, et al. American College of Surgeons Oncology Group (ACOSOG) Z0011: impact on surgeon practice patterns. Ann Surg Oncol 2012;19:3144e51. [27] Haffty BG, Hunt KK, Harris JR, Buchholz TA. Positive sentinel nodes without axillary dissection: implications for the radiation oncologist. J Clin Oncol 2011;29:4479e81. ~ ero A, Gime nez J, Merck B, V [28] Pin azquez C. Consensus meeting on selective biopsy of the sentinel node in breast cancer. Spanish Society of Senology and Breast Disease. Rev Esp Med Nucl 2007;26:176e80. [29] Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama F, et al. One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Cancer Res 2007;13: 4807e16. [30] Bloom H, Richardson WW. Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer 1957;11:359e77. [31] Ohi Y, Umekita Y, Sagara Y, Rai Y, Yotsumoto D, Matsukata A, et al. Whole sentinel lymph node analysis by a molecular assay predicts axillary node status in breast cancer. Br J Cancer 2012;107:1239e43. [32] Osako T, Iwase T, Kimura K, Horii R, Akiyama F. Sentinel node tumor burden quantified based on cytokeratin 19 mRNA copy number predicts non-sentinel node metastases in breast cancer: molecular whole-node analysis of all removed nodes. Eur J Cancer 2013;49:1187e95. rez-Hoyos S, Ramos M, Sancho M, Xercavins J, [33] Espinosa-Bravo M, Sansano I, Pe et al. Prediction of non-sentinel lymph node metastasis in early breast cancer by assessing total tumoral load in the sentinel lymph node by molecular assay. Eur J Surg Oncol 2013;39:766e73. [34] Peg V, Espinosa-Bravo M, Vieites B, Vilardell F, Antúnez JR, de Salas MS, et al. Intraoperative molecular analysis of total tumor load in sentinel lymph node: a new predictor of axillary status in early breast cancer patients. Breast Cancer Res Treat 2013;139:87e93. [35] Latosinsky S, Berrang TS, Cutter CS, George R, Olivotto I, Julian TB, et al. CAGS and ACS evidence based reviews in surgery. 40. Axillary dissection versus no axillary dissection in women with invasive breast cancer and sentinel node metastasis. Can J Surg 2012;55:66e9.