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Background Sentinel lymph node (SLN) biopsy is a widely used diagnostic procedure in the management of early breast cancer. When SLN is free of metastasis, complete axillary dissection may be skipped for staging in clinically N0 patients, allowing a more conservative procedure. Histological tumor features that could reliably predict SLN status have not yet been established. Since the degree of tumor lymphangiogenesis and vascularization may theoretically be related to the risk of lymph node metastasis, we sought to evaluate the relationship between lymph vessel invasion (LVI), lymphatic microvascular density (LVD), microvascular density (MVD) and VEGF-A expression, with SLN status and other known adverse clinical risk factors. Methods Protein expression of D2–40, CD34, and VEGF-A was assessed by immunohistochemistry on paraffin-embedded sections of primary breast cancer specimens from 92 patients submitted to SLN investigation. The presence of LVI, the highest number of micro vessels stained for D2–40 and CD34, and the protein expression of VEGF-A were compared to SLN status, clinicopathological features and risk groups. Results LVI was detected in higher ratios by immunostaining with D2–40 (p < 0.0001), what would have changed the risk category from low to intermediate in four cases (4.3%). There was no association between LVI and other angiogenic parameters determined by immunohistochemistry with SLN macrometastases, clinical features or risk categories. Conclusion Assessment of LVI in breast carcinoma may be significantly increased by immunostaining with D2–40, but the clinical relevance of altering the risk category using this parameter may not be advocated according to our results, neither can the use of LVI and LVD as predictors of SLN macrometastasis in early breast cancer.

BackgroundMetastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis.Methodology/PrincipalFindings Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process.Conclusions/SignificancePotential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development. published

In some patients, the radiocolloid used to perform sentinel lymph node biopsy (SLNB) for breast cancer appears in a number of lymph nodes and in different levels of the axilla. Most positive sentinel lymph node specimens (SLNSs) removed during SLNB are identified in level I of the axilla and within the first 4 SLNSs. Our objective was to verify the staging accuracy of harvesting only the first 4 SLNSs and to determine the relevance of SLNSs that reside in level II of the axilla. A prospective database documenting the method of identification, radioisotope count, order of retrieval, and axillary level of SLNSs from 893 SLNBs was analyzed. A median of 2 SLNSs (range 1–9) were removed per patient. More than 4 SLNSs were found in 8.0%. All SLNSs harboring the largest nodal metastases were identified within the first 4 harvested. Twenty-one percent (184 of 870) of patients had level II SLNSs; 4.9% (9 of 184) were positive. When SLNSs were positive in both levels I and II, the nodal metastases were always of greater or equal size in the level I nodes. Only one patient (0.5%) had a positive level II SLNS macrometastasis (>2 mm, pN1), with a negative level I SLNS, but it was the hottest node and was removed first. Removal of more than the first 4 hottest SLNSs does not improve staging accuracy. Level II nodes can be ignored if a hotter level I SLNS is first identified.

Background: Total tumoral load (TTL) in the sentinel lymph nodes (SLN) assessed by the OSNA assay is a new variable that is able to predict the likelihood of more axillary metastasis. Compared with the number of positive SLNs, the TTL is independent of the number of metastatic SLNs and a better predictor of further nodal metastasis. Although establishing specific cutoff-points of the TTL can be questionable because they may change in the future and increasingly become more patient specific, we have seen that TTL Methods: This is a retrospective cohort study of 145 consecutive patients with cT1-T2 invasive breast cancer with ultrasonographically node-negative treated between April 2010 to April 2013, where there is at least one positive SLN assesed by OSNA. We design a prediction tool based on the TTL results determined by OSNA to calculate the likelihood of not finding more positive non-SLN (http://www.vhebron.net/es/calculadora-risc-metastasi-2). Our group have demonstrated that TTL=15.000 could be a good cut off by its high NPV and sensitivity, 85.5% and 76.7%, respectively. Results: A total 325 SLNs were removed with a mean of 2.24. The type of SLN metastasis were macrometastasis in 85 patients (58.6%) and micrometastasis in 60 patients (41.4%). When considering patients with number of positive SLNs, of 109 patients with ≤2 positives SLNs and an cALND, 17 patients (22%) had non-SLN metastasis; and of 5 patients with ≥3 SLN, four (80%) had non-SLN metastasis. Taking the TTL results, of 51 patients with TTL Of 55 patients with TTL ≥15.000 copies/µL (with 1 or 2 positives SLNs) with cALND, 20 patients (36%) had non-SLN metastasis and there were a media of 2.5 non-SLN metastasis (range 1-8). Of 34 patients (62%) with one positive SLN with TTL ≥15.000 copies/µL, 12 patients (35%) had non-SLN metastasis; and of 21 patients with two positive SLN with TTL ≥15.000 copies/µL, eight patients (38%) had non-SLN metastasis. Conclusion: The total tumor load obtained by OSNA predicts non-SLN metastasis independent of the number of positive SLNs. The assess of the TTL in our cohort of patients show that patients with one or two positives SLNs with high TTL had high likelihood of non-SLN metastasis greater than 27% of Z0011 criteria. Prospective studies are needed to determine the clinical impact of this variable in patients outcome in the management of patients with SLN assesed by OSNA. Citation Format: Martin Espinosa-Bravo, Francesc Pérez-Ceresuela, Sebastian Diaz-Botero, Vicente Peg, Isabel T Rubio. Total tumoral load as a prediction tool of non-sentinel node metastases in patients with early breast cancer and positive sentinel lymph node assesed by OSNA [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-17.

Background: Intra-operative sentinel node biopsy (SNB) is performed for clinically node negative invasive carcinoma (IC). Despite a negative result for metastasis from rapid frozen section (FS) diagnosis of intra-operative SNB, some cases may be diagnosed positive when permanent sections (PS) are analyzed (i.e., false negative). In order to reduce the false-negative rate, it is necessary to determine why macrometastases measuring greater than 2 mm cannot be accurately diagnosed using frozen specimens. The aim of this study was to compare the pathological characteristics of false-negative and positive cases of macrometastasis using FSs. We also reviewed whether there were differences in postoperative prognoses between positive and false-negative cases. Methods: We retrospectively reviewed 1632 intra-operative SNBs collected from 2008 to 2011 at St. Luke’s International Hospital, Tokyo, JAPAN. Of these, 980 patients had undergone surgery for the treatment of IC without neoadjuvant chemotherapy. Lymph nodes were sectioned every 2 mm and examined. For FSs, we performed hematoxylin and eosin (HE) staining, and for PSs, we used HE and cytokeratin (AE1/AE3) staining. Image J (NIH Image, Bethesda, MD, USA) was used for assessment of the metastatic area of lymph nodes. Micro- and macrometastases were defined as metastatic lesions measuring between 0.2 and 2 mm or measuring 2 mm or more, according to TMN classification. Results: Using FSs, we identified 104 patients (10.6%) who were positive for macrometastasis, 16 patients (1.6%) who were positive for micrometastasis, and 860 patients (87.8%) who were negative for metastasis. Of the negative cases, the result was changed to micrometastasis in 37 cases (4.3%) and to macrometastasis (false-negative cases) in 14 cases (1.6%). Ten of the 14 patients did not have additional axillary lymph nodes dissection. The mean age of the patients was 55.1 ± 3.1 years for false-negative cases and 52.3 ± 1.2 years for positive cases. In terms of histological features, there were statistically significant differences between false-negative and positive cases for invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), and IDC plus ILC (p < 0.05). There were no statistically significant differences between false-negative and positive cases in terms of the total number of slices for lymph nodes (8.4 ± 1.6 vs. 9.7 ± 0.6, respectively; p = 0.41) but tendency in the number of slices for metastases (2.4 ± 1.2 vs. 4.8 ± 0.5, respectively; p = 0.07). The maximum area ratio of metastasis was significantly lower in false-negative cases than in positive cases (3.0% ± 0.1% vs. 20.0% ± 0.2%, respectively; p < 0.05). There was no significant difference in disease-free survival between false-negative and positive cases (p = 0.43). Conclusions: Although it is necessary to reduce false-negative cases, no difference in prognosis was detected in our study. False-negative cases had lower metastasis area ratios than positive cases diagnosed using rapid diagnosis of intra-operative SNBs, and ILC is known to be high in histological features. Therefore, using AE1/AE3 stain together with rapid diagnosis of intra-operative SNBs may lower the false-negative rate when a diagnosis includes ILC in pre-operation analysis. Citation Format: Eriko Abe, Naoki Hayashi, Toshinao Onoda, Yang Yang, Mieko Uno, Hideko Yamauchi, Sachiko Ohde, Koyu Suzuki. Comparison between positive and false-negative sentinel lymph nodes in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-32.

Background Axillary lymph node dissection (ALND) may not be necessary in women with breast cancer who have micrometastasis in a sentinel node (SN), owing to the low risk of non-SN (NSN) involvement. The aim of this study was to identify a subgroup of women with a micrometastatic SN and a negligible risk of positive NSNs in whom ALND may be avoided. Methods Some 237 of 241 women with a macrometastatic SN and 122 of 138 with a micrometastatic SN underwent completion ALND and were compared with respect to NSN involvement. The 122 patients with SN micrometastasis were further analysed to determine factors that could predict the risk of positive NSNs. Results A total of 121 (51·1 per cent) of 237 women with SN macrometastasis had positive NSNs compared with 22 (18·0 per cent) of 122 with SN micrometastasis (P < 0·001). Multivariate analysis showed that size of SN micrometastasis (odds ratio 3·49 (95 per cent confidence interval (c.i.) 1·32 to 9·23); P = 0·012) and presence of lymphovascular invasion (odds ratio 0·23 (95 per cent c.i. 0·05 to 1·00); P = 0·050) were significantly associated with positive NSNs. SN micrometastasis less than 0·5 mm in diameter combined with absence of lymphovascular invasion was associated with an 8·5 per cent risk of NSN involvement. Conclusion Size of micrometastasis and presence of lymphovascular invasion were significantly related to the risk of finding additional positive axillary lymph nodes when the SN contained only micrometastasis.

Fundamento. La extirpación del ganglio centinela es una técnica quirúrgica muy extendida cuyo objetivo es evitar el vaciamiento axilar en pacientes con cáncer de mama en estadios iniciales. La determinación de la infiltración tumoral puede ser evidente en caso de macrometástasis (>2mm), o difícil de detectar en las micrometástasis (2mm, 5 fueron micrometástasis

Aims: Because sentinel lymph node (SLN) biopsy for breast cancer has become well established, one of the challenges now is to determine which patients require a completion axillary dissection following a positive SLN biopsy. Methods: A prospective database of patients who underwent SLN biopsy for invasive breast cancer from July 1999 to November 2002 (n = 180) was analysed. Fifty four patients (30%) had one or more positive SLN, and all underwent a completion axillary dissection. This subgroup was further analysed to delineate which factors predicted non-SLN metastasis. Results: Twenty six of the 54 patients with a positive SLN had additional metastases in non-SLNs. Significant variables that predicted non-SLN metastasis included extranodal extension (odds ratio (OR), 17.399; 95% confidence interval (CI), 1.69 to 178.96) and macrometastasis within the SLN (OR, 6.985; 95% CI, 1.291 to 37.785). Conclusions: In patients with invasive breast cancer and a positive SLN, extranodal extension or macrometastasis within the SLN were both independent predictors of non-SLN involvement.

Background Axillary lymph node dissection (ALND) is important for improving the prognosis of patients with node-positive breast cancer. However, ALND can be avoided in select micrometastatic cases, preventing complications such as lymphedema or paresthesia of the upper limb. To appropriately omit ALND from treatment, evaluation of the axillary tumor burden is critical. The present study evaluated a method for preoperative quantification of axillary lymph node metastasis using positron emission tomography/computed tomography (PET/CT). Methods The records of breast cancer patients who received radical surgery at the Gifu University Hospital (Gifu, Japan) between 2009 and 2014 were reviewed. The axillary lymph nodes were preoperatively evaluated by PET/CT. Lymph nodes were dissected by sentinel lymph node biopsy (SLNB) or ALND and were histologically diagnosed by experienced pathologists. The maximum standardized uptake value (SUVmax) was measured in both the axillary lymph node (SUV-LN) and primary tumor (SUV-T). The SUV-LN/T ratio (NT ratio) was calculated by dividing the SUV-LN by the SUV-T, and the efficacies of the NT ratio and SUV-LN were compared using receiver operating characteristic (ROC) curve analysis. The diagnostic performance was also compared between the techniques with the McNemar test. Results A total of 171 operable invasive breast cancer patients were enrolled, comprising 69 node-positive patients (macrometastasis (Mac): n = 55; micrometastasis (Mic): n = 14) and 102 node-negative patients (Neg). The NT ratio for node-positive patients was significantly higher than in node-negative patients (0.5 vs. 0.316, respectively, P = 0.041). The NT ratio for Mac patients (0.571) was significantly higher than in Mic (0.227) and Neg (0.316) patients (P

Conflicting results were obtained by various research groups using the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells circulating in peripheral blood. Whereas 100% positivity was initially reported for stage IV patients, more recent investigations reported positive detection rates between 30% and 50% in patients with disseminated melanoma. While the high detection rate initially reported in metastatic melanoma may be explained by contamination problems, methodological differences in different steps of the technical procedure of RT-PCR may account for the differences reported in more recent examinations. Major differences may result from the kind of blood preparation, the RNA isolation method, the kind of RT enzyme used, and the gene targeted by PCR primers. In our experience, blood purification by a Ficoll gradient increased melanoma cell detection rates compared to RNA extraction from total blood or after erythrocyte lysis. Amplification of MelanA in addition to tyrosinase resulted in a 30% enhanced sensitivity of melanoma cell detection compared to amplification to tyrosinase alone, whereas gp100/pMel17 and MUC18 gene products were already detected in blood from nonmelanoma patients. These findings are in agreement with those of other groups. Currently, an increase in the sensitivity for detection of circulating tumour cells to more than 50% of patients with disseminated melanoma seems to be unlikely. It is interesting that between 15% and 30% positive results and sometimes more have already been obtained from patients with primary melanoma. So far, there is no data for judging the prognostic significance of the detection of circulating tumour cells in patients without clinically recognisable metastases. Our limited experience shows that staging examinations in these patients reveal no proof of macrometastasis. Therefore, it is presently unclear whether these positive findings are associated with long-term prognosis or if they merely reflect false positive findings in this highly sensitive RT-PCR technique.

Tumor markers for metastatic melanoma have, so far, been infrequently used. Although there is good evidence from several studies that protein S-100B serum measurement could serve as a valid marker for micro- and/or macrometastasis, the S-100B-LIAmat test has not been established as part of national and international recommendations for the care of melanoma patients. Future studies should focus on early detection of metastatic disease with S-100B compared to routine clinical, laboratory and technical evaluations. Furthermore, preliminary, interesting results on the predictive value of S-100B for the monitoring of metastatic patients during treatment should be verified in multicenter trials. The same considerations are true for melanoma-inhibitory activity (MIA), a new and interesting tool for the detection of metastatic melanoma. However, available data on the clinical use of on the MIA-ELISA are too limited to speculate on an even higher sensitivity and specificity than reported for S-100B.

Compared to primary tumor formation and invasion, relatively little is known about later stages of the metastatic cascade, including the seeding and colonization of distal tissues. To visualize these late steps in metastasis, we have generated an autochthonous mouse model of pancreatic ductal adenocarcinoma in which all pancreatic epithelial cells are genetically labeled with yellow fluorescent protein (the KPCY model). This system allows for the detection of single seeded tumor cells in distant organs, as well as micrometastasis and larger lesions. We have assessed the size distribution of metastatic lesions in two frequently colonized organs, the liver and lung, and found that single cells and clusters consisting of less than ten cells represent the majority of lesions in both tissues; however these small lesions only progress to gross metastasis in the liver, not the lung. We have previously used the KPCY model to study the dynamic process of epithelial-mesenchymal transition (EMT) and its involvement in invasion and dissemination from the primary tumor. We have extended these studies to assess the role of EMT and the reverse phenomenon, mesenchymal-epithelial transition (MET), in seeding and colonization at metastatic sites. Our results suggest that while EMT is favored for entry into the circulation, there may be selection for epithelial tumor cells or MET at the metastatic site. Additionally this model allows us to investigate the parallel evolution of metastatic lesions, from single cells to macrometastasis, and the associated metastatic microenvironment. Citation Format: Nicole M. Aiello, David L. Bajor, Robert H. Vonderheide, Ben Z. Stanger. Lineage labeling elucidates the natural history of metastatic colonization. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR14.

86 Background: Sentinel lymph node biopsy (SLNB) is not routinely recommended for women diagnosed with ductal carcinoma in situ (DCIS); however, when the pathologist cannot rule out microinvasion on core biopsy (CB), the surgeon must decide whether to perform a SLNB at the time of surgical excision. Up to 10% of patients with T1mi are found to have breast cancer cells in the axillary lymph nodes, but less than 2% contain macrometastasis making the utility of SLNB in this setting unclear. Methods: The University of Chicago pathology database was queried for patients whose CB showed DCIS suspicious for microinvasion (Smic) or definite microinvasion (Mic) from 2000 to 2012. We analyzed histology, imaging, nodal status, core needle size, and the use of myoepithelial immunohistochemistry (IHC) markers to identify microinvasion. Results: We identified 60 women with Smic and 19 women with Mic on CB. In the Smic group, 33% had infiltrating ductal carcinoma (IDC) in the surgical specimen (35, 30, 30, 1 and 0% for T1mi, T1a, T1b, T1c, and T2 respectively compared to 58, 21, 0, 10.5 and 10.5% in the Mic group). A SLNB was performed at the initial surgery in 38/60 (63%) of Smic and in all Mic biopsies; they were positive in 3/42 (7%) and 2/19 (11%) respectively (p=0.64). When N1mic was excluded, the incidence of macroscopic nodal disease was 1/42 (2.4%) and 1/19 (5.3%) p=0.53. Of those with Smic, we observed a higher proportion of IDC associated with a lesion size ≥ 14 mm on imaging (75% versus 15% for < 14 mm, p

Physiologically relevant animal models of micrometastasis are lacking; yet micrometastasis activation leading to macrometastasis and/or recurrent tumors is a major cause of cancer mortality and treatment resistance. Tumor cell implantation metastasis models typically use large numbers of tumor cells and immune compromised rodents together with removal of primary tumors to maximize the number, size and speed of macrometastases. Genetic and chemical/physical tumorigenesis models are often inconveniently slow and do not provide consistent metastasis. In order to develop a platform for investigating the biology of micrometastasis and for drug development to target micrometastases, we developed a model to study this phenomenon in vivo. The 4T1 triple negative syngeneic breast cancer model was chosen without resection of the primary tumor. A cell titration from 100,000 to 500 4T1-GFP tagged cells was first completed, with cells implanted into fat pad 3 of immune proficient 8 wk female BALB/c mice. Implantation of 15,000-100,000 cells resulted in 90-100% primary tumor take by 7 days; a small number (less than 15) micrometastases into the lungs by 2 wk; 70-90% of the animals having macrometastases (as evidenced by a distinct blood supply using tetrarhodamine labeled dextran) by 4 wk; and with most animals becoming moribund by 5 wk. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30-250 micrometastases in the lung (with many animals having 5-10 brain micrometastases) 2 wk post-implantation; with the first small macrometastases present at 4 wk; with most (80%) animals having macrometastases at 5 wk and animals becoming moribund by 6 wk post-implantation. With animals seeded with 500-1500 cells, the time of micrometastasis to macrometastasis conversion and animal survival could be prolonged, though primary tumor take was less (40-60%) than with 7,500 cells. Finally, this model using 7,500 cells was used to test the efficacy of a first-line chemotherapeutic agent for breast cancer, docetaxel, given at its maximal tolerated dose (MTD; 30 mg/kg weekly). Although primary tumor growth could be significantly reduced by docetaxel (P Citation Format: Michael A. Ihnat, Lora C. Bailey-Downs, Jessica E. Thorpe, Bryan C. Disch, Anja Bastian, Taleah C. Farasyn, Paul J. Hauser, Robert E. Hurst. Development and characterization of a natural model of triple negative breast micrometastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1389. doi:10.1158/1538-7445.AM2013-1389

Introduction: Intra-operative assessment of sentinel nodes (SLNs) allows immediate completion axillary dissection (cALND) in breast cancer patients. Molecular assessment such as one-step nucleic acid amplication (OSNA) promises greater sensitivity and provides a more accurate quantitative assessment than traditional methods. Our unit policy is to proceed to cALND in patients with macrometastases but not for micrometastases. However, evidence of upstaging has led us to seek to raise the threshold for proceeding to cALND. The CK19 mRNA copy number is an expression of the metastatic burden in the SLN and may be related to the presence of additional disease in the cALND. Since the original copy number threshold between micro (250–5000 copies/microliter) and macrometastasis (>5000 copies/microliter) was based on few patients and serial pathological sections, we investigated the mRNA copy number in patients with and without additional disease in the cALND. Methods: All patients in our unit undergo pre-operative axillary ultrasound with fine needle aspiration cytology of any suspicious nodes. Those with malignant cytology proceed directly to ALND. Radiologically and cytologically node negative patients undergo sentinel lymph node biopsy (SLNB) and OSNA. Electronic records of consecutive patients with invasive breast cancer undergoing SLNB with OSNA from August 2011 to March 2012 were retrospectively reviewed. Two parameters of mRNA copy number were examined: Copy number of the highest copy number SLN and the summed copy numbers of all positive SLNs. Their relationship to the presence of further disease in the axilla was examined using Student's t test. Results: Of 201 SLNBs, 45 (22%) had macrometastasis-positive OSNA and therefore underwent cALND (1 patient declined). Twenty patients (45%) had no further positive nodes (a negative cALND) with a total axillary metastatic burden of 1–2 in 11–27 nodes. Twenty four (55%) showed further disease (a positive cALND) with a burden of 2–20 in 9–30 nodes, including the SLNs. There was no significant difference in tumour size or grade between patients with additional positive nodes in the cALND compared with those with no further disease. There was no significant difference in the copy number of the highest copy number positive SLN (p = 0.44) or in the summed copy number of all positive SLNs (p = 0.36) between the cALND positive and negative groups. Conclusion: OSNA CK19 mRNA copy number does not correlate with the cALND metastatic burden. Therefore, raising the copy number threshold may be too simplistic as a method to better select patients with high probability of a positive cALND. A predictive model will be derived based on multivariate analysis of the larger patient population (>400 patients) that will have undergone SLNB with OSNA by the time of SABCS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-11.

Background: Large multi institutional studies have pointed that previous surgical resection of breast tumours before axillary sentinel node detection (ASLND) was the main criteria of failure of this technique. Screening campaigns provide small tumours and despite efforts to obtain a diagnosis of early breast cancer, this is not always obtained, due to small tumours or false negative results of micro biopsies. The aim of our series was to assess identification rates and false negative rates of ASLND after previous surgical resection of breast tumours. Material and Methods: In a prospective multi institutional setting (14 multidisciplinary teams), we have included patients with a previous breast tumour surgical resection for the diagnosis of infiltrative breast adenocarcinoma. Patients with only a core biopsy and no surgical removal of the tumor before axillary surgery were not included. Each patient underwent a secondary surgical procedure for ASLND and axillary lymphadenectomy, and sometimes a breast secondary surgical procedure for margins. ASLND was performed with the combined method, with blue dye and technetium. Pathology was performed with serial sectioning, eosin safron and immune histo chemistry (IHC). Results: From July 2006 to November 2011, 138 patients where included. The median tumor size was 9mm. Identification rate was 86% (118/138). A macrometastasis was found in 11 cases, in a sentinel node (9), or in a non sentinel node(2). False negative rate was 9% (1 false negative sentinel node with macrometastasis in non sentinel node from lymphadenectomy/11 cases with a macrometastasis in either a sentinel node or a non sentinel node). In 1 case a micrometastasis was found in a sentinel node through IHC, with a macrometastasis in a non sentinel node from lymphadenectomy. Without IHC or without the decision of performing a complementary lymphadenectomy in the case of micrometastasis, the false negative rate would have been 18%. Conclusions: After previous surgical resection of early breast cancer, ASLND remains feasible with a low identification rate of 86%, despite the use of the combined method. The False negative rate is acceptable with the use of IHC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-21.

Dear Editor, The observations of Remoundos et al. [1] are quite interesting. Effectively, there is not a direct relationship between presence of mRNA and expression of protein. In the first case of breast cancer in which we found lack of CK19 expression, absence of CK19 mRNA was confirmed by Sysmex S.L. by means of a RT-PCR, which confirmed that truly CK19-negative tumours can be found among those with a luminal phenotype and not only among those with a triple-negative phenotype. After terminating the study, we found five more cases lacking CK19 expression. For various reasons, we did not confirm the mRNA status of these tumours, but the one-step nucleic acid amplification (OSNA) assay was not performed and sentinel lymph nodes (SLN) were studied by means of standard procedures. Even though the proportion of breast cancers lacking CK19 mRNA is most likely lower than that of immunohistochemically CK19-negative tumours, we think it may be hazardous to perform the OSNA assay in cases lacking expression of CK19. In our paper [2], we reported a case of a luminal-A breast carcinoma expressing CK19 in only very few cells, both in the primary tumour and in the matched SLN macrometastasis. This metastasis probably would have been detected by the OSNA analysis, not as macrometastasis but with a diagnosis of micrometastasis or even as isolated cells. Dr. Remoundos affirms that they found on OSNA analysis a micrometastasis in a SLN from a patient with a breast cancer lacking CK19 expression. How do they know that that metastasis was a micrometastasis and not a macrometastasis?

Background The Oncotype Dx® recurrence score (RS) assay quantifies the risk of distant recurrence (rDR) and its use has increased despite the lack of prospective studies. Methods This is a cross-sectional study of consecutive patients (PTS) from our Institution with histologically confirmed invasive breast cancer (IBC) who underwent surgery with curative intent and in whom Oncotype Dx (ODx) was performed. The main objectives were to compare (1) the predicted rDR by RS and Adjuvant! (2) Risk allocation by RS and St Gallen Criteria and (3) the agreement between histological grading (HG) and RS. Results From October/2006 to April/2012, 105 PTS with ER positive IBC were evaluated. Median age: 55y. Sixty-eight (64.8%) were EC IA; axillary lymph node involvement was seen in 25 PTS (14 micro and 11 macrometastasis). The rDR by RS was low in 64 PTS (60.9%), intermediate in 34 (32.4%) and high in 7 (6.7%). According to Saint Gallen, 12 (12%), 68 (68%) and 20 PTS (20%) were classified as low, intermediate and high risk, respectively, among 100 classifiable PTS. There was no statistically significant agreement between risk allocation by RS and Saint Gallen criteria (Kappa coefficient = -0.043; p = 0.401). Ki-67 data was available for 89 PTS: Conclusions We found no statistically significant agreement between Oncotype Dx®, the Saint Gallen criteria, Adjuvant!Online, or Ki-67. However, a potential association between HG and RS was noted. The rDR may be overestimated by clinicopathological-based classifications Disclosure All authors have declared no conflicts of interest.

Objective To determine the identification and the percentage of the false negative of the sentinel nodes in patients with early breast cancer Hospital La Linea, during the period 2007 to 2010. Methods We collect fifty patients with early breast cancer, without clinical and ultrasonographic involvement of axillary nodes, from November 2007 to September 2010. We use the vital dye in the first twenty patients, and the combined technique with vital dye and albumin labeled with technetium 99 in the other thirty patients. The site of injection for patients who use blue dye was the subdermal near to the tumor, and to the patients who use the technetium was the periareolar technique. The sentinel node biopsy was examined during the surgery. The sentinel node was cut through its long axis and then with fine cuts of 250 microns, with freezing technique. The axillary dissection was completed in the first seventeen patients, and in the remaining patients we performed total axillary dissection if the sentinel node was positive for metastasis. Results Sentinel nodes were identified in 49 of 50 patients (98%), procedures. The only case where we did not identify the sentinel node was a patient in the combined technique. The percentage of nodes identified in the patients with vital dye was one sentinel node, and in the patients who use technetium the percentage was two sentinel nodes. The false negative rate was 8% (4 patients), in three of them we found a micrometastasis in the final examination, in the other one we found a macrometastasis. This cases of false negative in the sentinel node biopsy occurred at the beginning of the study. Pathologists acquired a learning curve for sentinel node examination, and practiced subsequently fine cuts in the intraoperative examination of the sentinel node. Conclusion This experience indicates that intraoperative examination of sentinel node biopsy is crucial for staging of the axilla. The rate of identification of sentinel node is excellent and with the use of the technetium the technique is more accurate. The improvement in the learning curve about the intraoperative examination of the sentinel node biopsy has decreased our rates of false negatives. Disclosure All authors have declared no conflicts of interest.

Background: In AJCC 7th edition, Stage I breast tumors have been subdivided into Stage IA and Stage IB; Stage IB includes small tumors (T1) with exclusively micrometastases in lymph nodes (N1mi), in order to facilitate further investigation. However, it is obvious that the current routine pathological examination has the limitation to detect micrometastasis, because it can examine only a part of lymph node. One-Step Nucleic acid Amplification (OSNA) assay was developed to overcome this limitation of the routine pathological examination. This assay can classify the patients into 4 categories, (++), (+), (+I), and negative. (++) and (+I) are theoretically regarded as high-volume metastasis corresponding to macrometastasis, and (+) as low-volume metastasis corresponding to micrometastasis. Since OSNA has the potential for standardization and semi-quantitative assay for evaluating the amount of tumor cells in a lymph node, we decided to apply a whole node into OSNA assay to maximize the semi-quantitative advantage in clinical settings. In the present study, we report the results of sentinel node (SN) examination with whole node OSNA assay. And we compare the results with the pathological examination to reveal the characteristics of OSNA assay. Patients and Methods: Data of 961 patients from January 2008 to March 2010 with clinically node-negative and pT1 breast cancer and having received SN biopsy with the radioactive tracer were evaluated. SNs were examined by whole node OSNA assay with Gene Amplification Detector RD-100i and Lynoamp®BC or 2mm-thick intraoperative frozen section with H&E staining for 442 and 519 patients respectively. We compared the performance of whole node OSNA assay and the pathological examination. And we performed two-population-z-test. Results: 1) SN positive rates of whole node OSNA assay and the pathological examination in T1 patients were 88/442 (19.9%, 95% CI; 16.3-24.0%) and 77/519 (14.8%, 95% CI; 11.9-18.3%), respectively. The difference was 5.1% (95% CI; 0.1-9.6%, p=0.046). 2) The populations of OSNA (++) or (+I) and macrometastasis were 51/442 (11.5%, 95% CI; 8.8-15.0%) and 55/519 (10.6%, 95% CI; 8.1-13.6%), respectively. There was no significant difference. On the other hand, the populations of OSNA (+) and micrometastasis were 37/442 (8.4%, 95% CI; 6.0-11.5%) and 22/519 (4.2%, 95% CI; 2.7-6.4%), respectively. The difference was 4.1% (95% CI; 0.9-7.0%, p=0.012). Conclusions: OSNA assay could detect more low-volume metastases corresponding to micrometastasis than the routine pathological examination. Therefore, it is suggested that OSNA can select accurate SN/micrometastasis cohort. Follow up of the patients is required to clarify the prognosis of OSNA (+) patients, and as the result, it may be possible to establish the new breast cancer staging system using OSNA results. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-02.

Abstract #3012 Background: Sentinel lymph node (SLN) status is highly predictive of overall axillary lymph node involvement. SLN positive patients(pts)with macro or micrometastasis require delayed axillary clearance. Intraoperative imprints have a good sensitivity for detection of macrometastasis but micrometastases are seldom diagnosed. Thus, we tested the intraoperative determination of axillary node metastasis according to the Rapid Gene Search Breast Lymph Node (BLN) Assay (Veridex) based on RT-PCR. Patients and methods:50 pts operated between September 17 and March 20 ,2008, had a SLN procedure. Lymph nodes were cut in 2 mm slices, and 1 out of 2 was examined with Rapid BLN Assay; the other one had intraoperative imprints and definitive histological assessement in paraffin-embedded sections including serial slices with hematoxilin eosine safran coloration and Cytokeratine AE1-AE3 immunochemistry. Results : 103 lymph nodes were examined with a median number of 2 SLN by pt. With Rapid BLN Assay, 12(9 pts) were positive and confirmed by histological interpretation on the other half for 11 (8 pts). 89 lymph nodes were negative (41 pts); in 1 (1 pt), isolated tumor cells (CTI) were detected in the other half at histological interpretation. Intraoperative imprints detected 4 macrometastases. One node declared positive at the intra-operative examination imprint corresponded to a granulomatous lesion after histological interpretation and Rapid BLN Assay was negative. Sensitivity was 31% for intraoperative imprints, 92% for Rapid BLN Assay and the histological examination of paraffin--embedded sections. The median time for intraoperative determination was 34 minutes for 1 SLN compared with 20 minutes for imprints. Conclusion: Given the high sensitivity of the rapid BLN assay, the excess duration of the procedure is acceptable for the surgeon. Furthermore, this procedure avoids a secondary axillary clearance in pts without intraoperative examination or with negative imprints and with metastases in SLN. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3012.

Abstract #207 BACKGROUND. Pure tubular carcinoma (PTC) is a favourable histologic subtype of breast cancer with a low rate of lymph node involvement. This study was initiated to assess the sentinel lymph node (SLN) detection rate and the rate of axillary involvement. METHODS. Inclusion criteria were a PTC (tubular component >90%) core biopsy diagnosed breast cancer smaller than 20 mm, and a homolateral clinically axilla free of nodal involvement. Patients underwent a SLND alone or with an Axillary level I-II Lymphadenectomy (AL). An AL was performed in the case of the learning curve, or in the case of SLN involvement – macrometastases, micrometastases or isolated cells - or in the case of mapping failure. RESULTS. From January 1999 to December 2006, 234 patients were selected from 9 French Comprehensive Cancer Centers. Median tumour size was 9.59 mm (1-22) . The SLN detection rate was 97.9% (229/234). The median number of SLN harvested was 2.15 (range 0-8). Respectively the frequency of macrometastasis - >2mm - was 2.6% (6/229), micrometastasis was 6.5% (15/229) and isolated cells was 0.8% (2/229). There were no false negative cases among the 24 patients with a systematic complementary axillary lymphadenectomy performed in the case of a learning curve. Axillary macrometastasis were found in non sentinel lymph nodes after completed axillary lymphadenectomy in 3 / 6 patients with macrometastasis, in 1/15 patients with micrometastasis and in 0/2 patients with isolated cells. There was no lymph node involvement for tumour size < 10mm. Pathological tumor size >10 mm was statistically correlated to the risk of axillary involvement (p=0.0223). CONCLUSIONS. This study confirms that in the case of PTC ≤ 20mm SLN detection is feasible and the risk of lymph node involvement very low. In the case of PTC Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 207.

Purpose: Sentinel lymph node (SLN) biopsy has replaced unnecessary axillary dissection in breast cancer surgery except when the nodes are positive for macrometastasis. But guidelines for isolated tumor cells (ITCs) found in SLNs has not yet been established and further study is ongoing. The goal of this study was to consider the implication of the isolated tumor cells found in the SLNs of Korean breast cancer patients. Methods: Between September 2003 and March 2008, 985 primary breast cancer patients underwent SLN biopsy. On reviewing the medical records, 81 patients were found to have ITCs in SLNs without macrometastasis or micrometastasis. ITCs were detected by serial sectioning and immunohistochemistry. Results: The mean number of detected SLNs was 3.5±1.7. Thirty three patients had multifocally distributed ITCs and 9 had ITCs in multiple SLNs whose N stage was N0 (i＋). Completion axillary dissection has been performed in 9 patients and 3 of them (33.3%) finally were found to be N1 or N1mi. Conclusion: The characteristics of ITCs are not clear yet and their prognostic value is still under investigation. Until the significance of ITCs found in SLNs become definite, axillary dissection should be more aggressively considered.

Tumor resection in papillary thyroid carcinoma (PTC) is often accompanied by lymph node (LN) removal of the central and lateral cervical compartments. One-step nucleic acid amplification (OSNA) is a polymerase chain reaction-based technique that quantifies cytokeratin 19 (CK19) messenger RNA copies. Our aim is to assess the value of OSNA in detection of LN metastases in PTC, in comparison with imprints and microscopic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue. A total of 387 LNs from 37 patients were studied. From each half LN, 2 imprints were taken and analyzed with hematoxylin and eosin (HE) and CK19 immunostaining. One half of the LN was submitted to OSNA and one half to FFPE processing and HE and CK19 staining. For concordance analysis, every single LN was considered as a case. A group of 11 cases with discordant results between OSNA and HE/CK19 FFPE sections were subjected to additional FFPE serial sectioning and HE and CK19 staining. We found a high degree of concordance between the assays used, with sensitivities ranging from 0.81 to 0.95, and specificities ranging from 0.87 and 0.98. OSNA allowed upstaging of patients from pN0 to pN1, in comparison with standard pathologic analysis. Identification of a metastatic LN with more than 15000 CK19 messenger RNA copies predicted the presence of a second LN with macrometastasis (5000 copies). In summary, the study shows that OSNA application in sentinel or suspicious LN may be helpful in assessing nodal status in PTC patients.