The prognostic impact of occult nodal metastasis in early breast carcinoma

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1 DOI /s CLINICAL TRIAL The prognostic impact of occult nodal metastasis in early breast carcinoma Daehoon Park Æ Rolf Kåresen Æ Bjørn Naume Æ Marit Synnestvedt Æ Elsa Beraki Æ Torill Sauer Received: 1 October 2008 / Accepted: 3 February 2009 Ó Springer Science+Business Media, LLC Abstract The clinical relevance of isolated tumor cell (ITC: B0.2 mm) and micrometastasis (MM:[ mm) in axillary lymph nodes (ALNs) remains unknown. The aim of this study was to determine their prognostic significance. A total of 295 patients considered as pn0 after routine histological assessment, were reevaluated with ten-level cytokeratin immunohistochemistry (IHC) and two-level hematoxylin-eosin sections. Survival rates, i.e. disease-free survival (DFS), distant disease-free survival (DDFS) and breast cancer specific survival (BCSS) were compared with those of reevaluated node-negative patients. A total of 84 patients (28%) had ITC/MM identified on IHC sections. ITC had no impact on survival at a median 8.2 years of followup, whereas MM showed a trend toward poorer DFS (P = 0.091, log rank) and DDFS (P = 0.066) and significantly reduced BCSS (P = 0.016). In multivariate analyses, detection of MM was an independent prognostic factor for DDFS (P = 0.025) and BCSS (P = 0.01) in adjuvant untreated patients. Micrometastases (MMs) in axillary lymph nodes have prognostic impact. This was not found for ITC. This finding supports the use of systemic adjuvant therapy in patients with MM. D. Park (&) E. Beraki T. Sauer Department of Pathology, Oslo University Hospital, University of Oslo, Kirkeveien 166, 0407 Oslo, Norway daehoon.park@ulleval.no R. Kåresen Department of Surgery, Oslo University Hospital, University of Oslo, Oslo, Norway B. Naume M. Synnestvedt Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway Keywords Occult metastasis Axillary lymph node Invasive breast cancer Survival Prognosis Introduction The axillary lymph node (ALN) status is the most powerful predictor of survival and has clinical implications for adjuvant therapy in breast cancer patients [1]. Standard complete axillary lymph node dissection (ALND) used to be the traditional approach to assess the status of axillary lymph node metastasis. During the past decade, however, sentinel lymph node biopsy (SLNB) has replaced ALND as a procedure for providing accurate staging of the axilla with a reduction of the associated morbidity, such as lymphedema, seroma, limitation of shoulder motion, pain and paresthesia [2, 3]. In the sixth edition of the American Joint Committee on Cancer (AJCC) Staging Manual [4], micrometastases (MMs) are defined as tumor deposits from 0.2 to 2.0 mm in size. Unlike micrometastases, isolated tumor cells (ITCs) are defined as single tumor cells or small clusters of cells that are B0.2 mm in size and usually show no histological evidence of malignant activity. MM and ITC are often grouped together as occult metastasis. In most institutions patients with MMs are recommended ALND and systemic adjuvant therapy as for node-positive patients, while those with ITCs are classified as node-negative patients (pn0i?) and, depending on the primary tumor characteristics, may not receive adjuvant therapy. A number of studies have re-evaluated the lymph nodes of breast cancer patients that were considered negative by initial routine histological assessment using hematoxylin and eosin (H&E) staining [5 14]. They found that 9 32% of previously node-negative cases had occult tumor cell

2 deposits after review, using various methods such as serial sectioning and/or immunohistochemical (IHC) staining [5]. Despite an overall good prognosis for node-negative patients, development of distant metastases is still a threat for this group of patients [15, 16]. Although some studies support the prognostic impact of occult tumor cell deposits in axillary lymph nodes [8, 9, 13, 14], others have not found such correlation [7, 10 12]. Susnik et al. demonstrated that distant metastases developed somewhat more frequently in the group of patients with ITCs than in those without tumor deposits [13]. Tan et al. also showed a prognostic significance of ITCs in term of disease free survival [14]. Several studies, however, have documented a correlation between nodal micrometastases (MMs) and prognosis, emphasizing the need for more aggressive therapy if MMs are present [8, 13, 14]. In contrast, other studies argue that the prognosis of MM patients is similar to the outcome of node-negative patients [12, 17]. Therefore, the impact of occult tumor cell deposits in axillary lymph nodes, whether it is MMs or ITCs, on clinical outcomes and therapeutic strategy is still under debate. In a smaller study, including patients from the same population as the present study [18], we reported a lack of covariance between the detection of micrometastases in lymph nodes and bone marrow suggesting independency of lymphatic and hematogenous spread. This observation is also supported by other authors [17, 19]. The primary aim of the present study was to determine the relation between ITC/MM in axillary lymph nodes and tumor dissemination and clinical outcome in a cohort of early breast cancer patients originally considered as pn0 after routine histopathologic examination. A secondary aim was to study covariance between lymph nodes and bone marrow micrometastasis. Materials and methods Patients Between May 1995 and December 1998, 920 consecutive women with invasive breast cancer, who underwent mastectomy or breast conserving surgery with ALND from hospitals in the Oslo region, were included in the Oslo Micrometastatic Breast Cancer Study. The patients underwent bone marrow aspiration and detection of disseminated tumor cells in bone marrow at surgery as previously published [20, 21]. Of these, 298 consecutive patients with lymph node negative status as determined by routine histopathological examination (1 2 H&E stained sections) treated at Ullevaal University Hospital and Norwegian Radium Hospital, were included in the current study. Three patients were excluded because final histology was ductal carcinoma in situ. A total of 295 patients were further evaluated in the study. Informed and written consent was obtained from all patients. All clinicopathological data were retrieved from the Oslo Micrometastatic Study database [21]. The characteristics of the study population and their specimens are summarized in Table 1. Two hundred Table 1 Characteristics of study population (N = 295) and their specimens Age (years) Median (range) 59 (28 90) B50 52 (17.6%) [ (82.4%) Adjuvant systemic therapy No 232 (78.6%) Yes 62 (21.1%) Unknown 1 (0.3%) Primary tumor type Invasive ductal carcinoma 217 (73.6%) Invasive lobular carcinoma 54 (18.3%) Others 24 (8.1%) Primary tumor grade (35.6%) (45.8%) 3 48 (16.3%) Unknown 7 (2.4%) Primary tumor size T1a-b 92 (31.2%) T1c 129 (43.7%) T (21%) Tx 12 (4.1%) Hormone receptor status Positive 233 (79.0%) Negative 53 (18.0%) Unknown 9 (3.1%) Vascular invasion Absent 244 (82.7%) Present 21 (7.1%) Unknown 30 (10.2%) BM status Negative 262 (88.8%) Positive 17 (5.8%) Unknown 16 (5.4%) HER-2 status Negative 251 (85.1%) Positive 7 (2.4%) Unknown 37 (12.5%) P53 status Negative 234 (79.3%) Positive 45 (15.3%) Unknown 16 (5.4%)

3 Table 1 continued Largest metastatic tumor size (mm) Neg 203 (68.8%) ITC (B0.2) 53 (18%) MM ( ) 31 (10.5%) [2.0 mm 8 (2.7%) IHC/HE staining IHC-/HE- 203 (68.8%) IHC?/HE- 51 (17.3%) IHC?/HE? 41 (13.9%) Number of positive lymph nodes (68.8%) 1 51 (17.3%) C2 41 (13.9%) Presence of occult metastases in LN No 203 (68.8%) Yes 92 (31.2%) and thirty two patients (79%) did not receive adjuvant systemic therapy. The rest of the patients received treatment according to the Norwegian guidelines at the time of diagnosis [21, 22]; (a) chemotherapy (cyclophosphamide 600 mg/m 2, methotrexate 40 mg/m 2, 5-fluorouracil 600 mg/m 2 3qw (CMF), 9 cycles) followed by tamoxifen if estrogen receptor (ER) and/or progesterone receptor (PgR) positive and age \55 years, (b) only CMF if ER and PgR negative and age \65 years, (c) only tamoxifen if ER and/or PgR positive primary tumors and age C55 years. Follow-up for median 98 months (range months) included clinical examination at 6 12 months intervals with annual mammography. Further diagnostic work-up was performed only if the patients had symptoms or signs of progression. Immunohistochemistry of the ALN The formalin-fixed, paraffin embedded node tissue was sectioned serially perpendicular to the long axis at 100 lm intervals up to 10 levels, with 4 lm thick sections obtained from each level. All ten slides were subjected to IHC staining. One additional section from each node was made from the third and tenth level for H&E staining. For immunostaining slides were fixated in incubator min at 56 C and overnight at 37 C. The slides were deparaffinized twice in xylene and rehydrated through descending graded alcohols to water, and then were placed in the Benchmark XT instrument (Ventana Medical Systems, Inc., Tucson, AZ) for IHC staining with the two-pancytokeratine antibodies AE1 and AE3 (Ventana Medical Systems, Inc. Tucson AZ, USA). Immunostained cells were visualized with DAB detection kit after dehydration. Positive controls with known LN metastases were included. All slides were examined by the primary investigator (DP) using a light microscope. The SLNs were recorded as positive when cytokeratin positive cells were found. Cells were considered to be occult metastases when they were identified on the H&E or IHC sections. According to the AJCC classification, metastases more than 2.0 mm in diameter are categorized as macrometastases, groups of tumor cells larger than 0.2 mm but B2.0 mm in diameter as micrometastases (MM) and tumor deposits less than or equal to 0.2 mm in greatest dimension as ITC. Subsequently, the absence or presence of metastatic cells on H&E sections (from level 3 to 10) was also registered. Slides with cells that were difficult to classify, were reviewed by a pathologist (TS). Analysis for ITC in bone marrow BM was aspirated from anterior and posterior iliac crests bilaterally (10 ml/site) and processed as described previously [20]. Briefly, mononuclear cells were collected, followed by preparation of cytospins and immunocytochemical staining of the slides (consisting of cells) with anti-cytokeratin mabs (AE1AE3) and visualization of cytokeratin-positive cells with APAAP technique. The results of these analyses have been published in detail previously [20, 21]. The ITC-status in BM from the patients included in the present study was retrieved from the Oslo Micrometastatic Study database [21]. ITC-positivity was defined as C1 tumor cell-compatible cytokeratinpositive cells in the specimen. Statistical analysis Clinical end points in the survival analysis were: Disease free survival (DFS), distant disease free survival (DDFS) and breast cancer specific survival (BCSS), which was calculated from the date of the primary surgery to the date of the first relapse, systemic relapse or breast cancer-related death, and otherwise censored at the time of the last followup visit or at non-cancer related death. DFS was defined as absence of any event considered related to the primary cancer (e.g. axillary metastases, loco-regional relapse, ipsilateral carcinoma or distant metastasis), but excluding contralateral breast cancer and any other cancer. Metastasis to the skeleton, liver, lung or CNS was recorded as systemic relapse. Kaplan Meier survival curves were constructed and the survival rates were compared using the log-rank test. Uni- and multivariate analyses of the independent prognostic factors were performed using the Cox proportional hazards regression model (stepwise backward elimination). All the variables with a P-value equal to or less than 0.1 in the univariate analysis were included in the

4 multivariate analysis. To compare categorical variables, chi-square test or Fisher exact test were used. A P-value of \0.05 was considered statistically significant. All statistical analyses were performed using the statistical software SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Detection of occult metastases in ALN Occult metastases were categorized as either ITC (largest tumor cell cluster size B0.2 mm) or micrometastasis (largest tumor cell cluster size [0.2 to B2.0 mm), as illustrated in Fig. 1. Altogether, occult metastases, including ITC, MM and macrometastasis, were detected in 92 of 295 patients (31%), Isolated tumor cells (Fig. 1a b) were detected in 57% (53 of 92), whereas MM (Fig. 1c d) in 34% (31 of 92). In 9% (8 of 92) of the cases, macrometastases were found. Of the 53 cases with ITCs, 41 were seen by IHC only and were not detectable by H&E stain alone (77%). Of the 31 with MM, 10 were detectable by IHC stain alone (32%). All eight macrometastases were seen by both IHC and H&E stain. Seventeen of 31 (54.8 %) MMs appeared in the upper three levels. In the next three levels (4th 6th), additional 7/31 (22.6%) was detected, as compared to 7/31 (22.6%) in the last 4 levels (7th 10th). In 19 (61%) of the cases the MM appeared in more than one level. Among the 84 patients with ITC or MM, 73% had invasive ductal carcinoma (IDC), 24% had invasive lobular carcinoma (ILC) and 3% had other types, in accordance with the frequency distribution of these tumor subtypes in the cohort. Analyzing the ITC? and MM? separately, 66% of the ITC? was IDC, as compared to 84% of the MM?. The same figures for ILC were 28% for ITC? and 16% for MM?. Of the 61 IDCs, 51% (31 of 61) was detectable by IHC stain alone, whereas 85% (17 of 20) of the ILCs were detected by IHC only. Association between occult metastases in ALN and BM ITCs were found in BM in 17 of the 295 patients (6%). The correlation between ITC in BM and occult metastasis in lymph nodes are presented in Table 2. No significant association was found between BM or ALN occult metastases neither in the total material (P = 0.680, v 2 -test) nor in the 232 patients which did not receive adjuvant therapy (P = 0.480, Fisher s exact test). Histologic findings in the lymph nodes A total of 3,425 lymph nodes were examined from 295 patients. The number of removed nodes per patient ranged from 1 to 29 with a median of 11. The percentage of positive ALN in the ITC and MM groups is shown in Fig. 2. Of the 53 ITC? cases, 36 (67.9%) had metastatic tumor cells identified in only one Fig. 1 Isolated tumor cell (ITC) (a) and micrometastasis (MM) (c) detected by H&E, and ITC (b) and MM (d) detected by anticytokeratin IHC (AE1/ AE3). (magnification 9200)

5 Table 2 Comparison of ALN and BM status All patients (N = 295) Non adjuvant therapy (N = 232) ALN (-) ALN (?) P-value ALN (-) ALN (?) P-value ALN, Axillary lymph node; BM, bone marrow a Chi-square test b Fisher s exact test BM (-) 182 (65.2 %) BM (?) 11 (3.9 %) 80 (28.7 %) 6 (2.2 %) a 145 (65.6 %) 9 (4.1 %) 64 (29.0 %) 3 (1.4 %) b Fig. 2 Correlation between the size of the occult metastases (ITC/ MM) and the number of positive axillary lymph nodes. Total numbers of ITC and MM patients are 53 and 31, respectively lymph node, as compared to 23 of the 31 MM? patients (74.2%). Survival analyses Eight patients with macrometastases were excluded from the Kaplan Meier survival analyses. At a median observation time of 98 months (range months), 31 patients had developed relapse, of these 2 local and 29 systemic relapses. Fifteen patients had died of breast cancer. No significant difference in DFS, DDFS and BCSS was found between node-negative and ITC patients (Fig. 3), whereas MM patients showed significantly reduced BCSS in both total and no adjuvant therapy cases (P = 0.04 and P = 0.01, respectively) compared to those without evidence of occult metastasis to ALN (Fig. 3b, d). Patients with MM had a trend toward worse DFS (P = 0.08 and P = 0.09) (Fig. 3a, c) and DDFS (P = 0.06 and P = 0.07, not shown) in both all and adjuvant untreated patients, respectively. Uni- and multivariate analyses Univariate analysis of various prognostic factors, BM status and the occult metastasis status are listed in Table 3. Eight cases with macrometastases were excluded from the analyses. Among the remaining 287 patients, the analysis identified primary tumor grade, primary tumor size, hormone status and vascular invasion to be significant predictors of DFS, DDFS and BCSS, as well as MM for BCSS. In patients who did not receive adjuvant systemic therapy, the same parameters were associated with the same survival end points, except that MM was prognostic both for BCSS (Table 4) and DDFS (RR = 3.3, 95% CI: , P = 0.025). In addition, age was a significant prognostic factor for DFS but not for BCSS in both all and the non-adjuvant therapy patients. In multivariate analyses of all patients, age, primary tumor size and hormone receptor status were independent prognostic factors for DFS (Table 4) and DDFS (not shown). The detection of MM showed a borderline significance for DFS (Table 4) and DDFS (RR = 2.5, 95% CI: , P = 0.052). Histologic grade was the only independent predictor of BCSS. In the no adjuvant subgroup, vascular invasion was a significant prognostic factor for DFS (Table 4) and MM for DDFS (RR = 3.3, 95% CI: , P = 0.025), in addition to the parameters detected for all patients. Hormone receptor status and detection of MM was significant for BCSS (Table 4). Discussion The presence of regional lymph node metastases, together with tumor size and histological grade, has been the most important factors in the decision of which patients should have adjuvant therapy in the treatment of primary breast cancer. However, the clinical significance of occult metastasis detected in ALN of breast cancer patients remains controversial. Nonetheless, there is growing evidence that the detection of occult metastases in ALN could be associated with increased risk of recurrences and worse survival [23 26]. In our study, occult metastases, including both ITC and MM, but excluding macrometastasis, were identified in 84 of 295 previously routine node negative patients (28%) using cytokeratin-ihc and serial sectioning. Our 28% detection rate of ITC and MM in all reviewed cases is within earlier reported range [5]. About 68% of MMs were identified in both H&E and IHC sections while 32% where found by IHC alone. Susnik et al. [13] found

6 Fig. 3 The Kaplan Meier survival analyses [DFS (A&C) and BCSS (B&D)] of all patients (n = 287, 8 with macrometastasis excluded) and non-adjuvant therapy cases (n = 226, 6 with macrometastasis excluded) according to the presence/ size of occult metastases. The P-values using the log rank test are shown in the figures. No significant difference in the three survival rates (DFS and BCSS) was found between node-negative and ITC patients that all MMs were detectable by H&E serial section. They suggested that IHC facilitated the detection of ITC, but was not contributory in detection of MMs [13]. However, the usefulness of IHC analysis in SLN should be evaluated in relation to its added value to H&E sections in the detection of cancer cell deposits which have impact on the prognosis of un-treated patients and thus for the adjuvant treatment strategies. Use of methods with increased sensitivity for the detection of deposits without such clinical impact, is of no benefit for the patient and results in unnecessary costs. Our results show that ITC have no impact on survival compared with node-negative patients at a median 8.2 years of follow-up, whereas MM? patients showed a trend toward poorer DFS and DDFS and significantly reduced BCSS. MM-status was an independent prognostic factor for DDFS and BCSS in patients not receiving

7 Table 3 Univariate survival analyses in all patients (N = 287) and in patients without adjuvant therapy (N = 226) Variables DFS BCSS RR 95% CI P-value RR 95% CI P-value All patients (N = 287) Age (years) B [ Primary tumor type IDC ILC Primary tumor grade G G \ \0.001 Primary tumor size B2 cm [2 cm HR status Pos Neg \0.001 Vascular invasion Yes No of pos. LN C IHC/HE status IHC-/HE IHC?/HE IHC?/HE? BM status Neg Pos Largest metastatic tumor size Neg ITC (B0.2 mm) MM( mm) Detection of MM Yes Presence of occult metastases Yes Patients without adjuvant therapy (N = 226) Age (years) B [ Primary tumor type IDC ILC Primary tumor grade G G \ \0.001 Table 3 continued Variables DFS BCSS RR 95% CI P-value RR 95% CI P-value Primary tumor size B2 cm [2 cm HR status Pos Neg \0.001 Vascular invasion Yes No of pos. LN C IHC/HE status IHC-/HE IHC?/HE IHC?/HE? BM status Neg Pos Largest metastatic tumor size Neg ITC (B0.2 mm) MM ( mm) Detection of MM Yes Presence of occult metastases Yes IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; IHC, immunohistochemistry; HE, Hematoxylin-eosin; ITC, isolated tumor cell; MM, micrometastases; DFS, disease free survival; BCSS, breast cancer specific survival; RR, relative risk; CI, confidence interval adjuvant therapy, which in our opinion supports the use of systemic treatment in such cases. Contrary to this, Tan et al. found that the prognosis of patients with ITCs was worse than that of node-negative patients [14]. However, our results are consistent with the majority of such studies [8, 10, 13]. Although the observation time is relatively long (median 8.2 years) in our study, we cannot exclude that longer follow-up is needed for mature prognostic information to appear. However, the Kaplan Meier plots for ITC positive no adjuvant treated patients show no relapses at all during the last[65 months follow-up (Fig. 3), which may question the importance of additional follow-up. Furthermore, an automated screening method for the evaluation could improve the ITC detection rate even further. However, as the ITC detected by manual screening did not give prognostic information, a method that might

8 Table 4 Multivariate analyses in all patients (N = 287) and in patients without adjuvant therapy (N = 226) Variables DFS BCSS RR 95% CI P-value RR 95% CI P-value All patients (N = 287) Age (years) [ Not included a B Primary tumor grade G G \0.001 Primary tumor size B2 cm [2 cm Hormone status Pos Neg Vascular invasion Yes Detection of MM Yes Patients without adjuvant therapy (N = 226) Age (years) [ Not included a B Primary tumor grade G G Primary tumor size B2 cm [2 cm Hormone status Pos Neg Vascular invasion Yes Detection of MM Yes All the variables with a P-value equal to or less than 0.1 in the univariate analysis were included in the multivariate analysis (Cox proportional hazards regression model, stepwise backward elimination) a For age, the P-value for BCSS in the univariate analysis were more than 0.1 IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ITC, isolated tumor cell; MM, micrometastasis; DFS, disease free survival; BCSS, breast cancer specific survival; RR, relative risk; CI, confidence interval increase the sensitivity for detection of ITC, would probably not affect our results. Our study has, as many others [5 7, 9, 12, 23], shown that cases with MM were missed if IHC and/or serial sectioning were not used (10 of 31). As we have found that MM are clinically relevant, to miss detection of MM is unwanted. Using H&E staining, Madsen et al. [27] have suggested that the interval between the sections must be decreased to 200 lm, and 20 levels from each of a bisectioned lymph node must be investigated to detect micrometastasis with a 95% probability. Accepting such a labor-intensive method in the routine laboratory may, however, not be feasible. In our opinion, it may not be necessary to perform the serial sectioning with 100 lm interval as used in our study, because ITC equal to or less than 0.2 mm (200 lm) has no prognostic impact on the clinical outcomes. However, as the largest diameter of a tumor deposit could be in any direction, increased sectioning interval may have the chance of missing MM, but probably to a limited extent. We agree with Madsen et al. [27] that the interval between the sections could be increased up to 200 lm with only a low risk of missing MM. In a clinical perspective, it is still uncertain how many sections should be performed to have a satisfactory accuracy of MM detection. We found 55% of MM in the upper three levels, the rest was evenly distributed among the next levels (4 10) and there were no leveling off of the findings in the deeper levels. Thus it is probable that even more MM could have been detected if deeper sections had been examined. Based on our results and what is clinically feasible, however, we suggest at least five sections with 200 lm interval to be analyzed from each sentinel node. Another question is whether H&E stained sections still would be sufficient to detect most MMs. In the way we have carried out our study, 21/31 MM (68%) were found by H&E (all these also by IHC) while 10/31(32%) were found by IHC alone and were not seen in the H&E sections of these patients. As there were only two H&E sections (at 3rd and 10th level) and 10 IHC sections, we cannot know whether the MM detected by IHC alone also could have been detected by H&E. Further studies should address this, including serial sectioning for H&E and IHC taken adjacent to each other. It is reasonable to presume that IHC would be more important for infiltrating lobular carcinomas than for infiltrating ductal carcinomas as we, in accordance with Tan et al. [14], have found that ILC cases (38%) had a higher rate of ITC or MM than IDC cases (29%). Among patients with ITC or MM, 51% (31/61) was detectable by IHC stain alone for IDC compared to 85% (17/20) for ILC. ILC also comprised more occult ITCs in ALN than IDC (75 vs. 57%). This observation reflect the difficulty of detecting small groups or single lobular carcinoma cells in ALN using routine H&E stain with a single or two sections.

9 The findings could be explained by the fact that combinations of irreversible genetic and epigenetic events of E-cadherin gene locus in ILC, could lead to loss of E-cadherin expression, weakness of intercellular adhesion and thus a higher probability of metastasis in form of a single cell or small cluster in ILC as compared to IDC [28]. In contrast to Tan et al. [14], our results indicated no distinct importance of serial sectioning with IHC stain in terms of the impact of ITC on clinical outcomes, although occult ITC/MM in ILC cases were more detectable by IHC analysis alone than those of IDC. However, as the number of patients with ILC was small, we cannot exclude the possibility that ITC status may have different prognostic impact in IDC as compared to ILC. Our data support the assumption that lymphatic and hematogenous tumor spreads represent at least partially independent metastatic processes, as there was no significant association between positive ALN and BM ITCs in early breast cancer patients. However, the present investigation was composed of previously routine H&E node-negative patients and a small number of patients with positive BM status (6%). In contrast to our observation, Langer et al. [29] found a correlation between the SLN status and the presence of BM micrometastases in early breast cancer patients. In their study, where ITCs in the SLN were regarded as negative, a significant association between MM and BM positivity was found in both bivariate as well as multivariable analysis. Nonetheless, the percentage of non-concordance (SLN positive/bm negative 20% and SLN negative/bm positive 16.4%) was considerable. Despite the fact that ALN-status/BM-status is non-concordance in our study, the observation that MM in ALN has prognostic impact, does indicate that MM in ALN can be associated with hematogenous spread. The degree of concordance can be affected by the sensitivity of the method for detection of ITC in BM. However, the statement [17]that the diagnosis of all types of ALN involvement has therapeutic relevance because parallel hematogenous spread of tumor cells is likely to have occurred is in our opinion doubtful. Carter et al. [30] suggested that benign transport of breast epithelium into ALN after needle or surgical manipulation could be misinterpreted as local micrometastatic disease as well as an altered red blood cells and hemosiderin-laden macrophages. In our study, eight cases with morphologically uncertainty of tumor cells on IHC section were selected and stained with both macrophage marker CD68 and cytokeratin-ihc simultaneously. However, none of the cases had double staining (not shown). This would indicate that the cells in the ALN were of epithelial origin and not macrophages. However, it is not possible to conclude whether the cells are benign or neoplastic. Recent literature suggested that even if a sentinel node is positive and not accompanied by ALND, the risk of regional axillary nodal recurrence is only about 2% [31]. Fisher et al. [32] randomized patients with clinically negative axillary nodes to radical mastectomy, total mastectomy plus radiotherapy or total mastectomy alone and demonstrated that axillary treatment with either dissection or regional radiotherapy reduced axillary recurrences rates from 18.6 to 1 2%. However, there was no benefit of axillary treatment in terms of distant disease free survival. In the present study all patients were from pre-sentinel node biopsy era. We found that 74.2% of the MM patients had metastatic tumor cells identified in only one lymph node of a median number of removed lymph nodes of 11 (Fig. 2). When we find occult metastases in only one axillary lymph node, it may be reasonable to assume that the majority of these affected nodes are sentinel nodes. In accordance with Kuijt et al. [33], it might be acceptable to avoid ALND when only one positive sentinel node containing MM is identified. Another study [34], supporting this assumption, demonstrated that 250 (78.6%) of the 318 patients with SLN MM ([ mm in size) had no additional axillary node involvement, and SLN biopsy itself is sufficient to remove all micrometastases in the majority of situations. However, a possible impact on the survival of the approximately 25% having metastasis, located in other than the sentinel node containing MM, should be taken into consideration. Based on our study, we cannot know if such an avoidance of ALND would give eventual remaining tumor cell deposits in a non-sentinel node the possibility to develop and metastasize, as all the patient were primarily treated by ALN dissection. The possibility that X-ray [35] or even systemic treatment could compensate for not utilizing ALND in MM patients must also be taken into consideration: Randomized trials to evaluate this hypothesis are urgently needed. One is underway (EORTC 10981), which includes X-ray to the axilla for those that ALND is omitted [35]. In conclusion, we found that occult metastasis (MM) of [ mm in previously negative ALN by conventional H&E assessment, significantly affects clinical outcomes. This supports the use of systemic adjuvant treatment for patients with MM. 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