1. EVALUATION OF A MULTIPARAMETRIC SYSTEM ABLE TO PREDICT NON-SENTINEL LYMPH NODE STATUS IN BREAST CANCER PATIENTS WITH A MICROMETASTATIC SENTINEL NODE ASSESSED BY THE ONE STEP NUCLEIC ACID AMPLIFICATION (OSNA) ASSAY Simonetta Buglioni, Marcella Mottolese, Beatrice Casini, Enzo Gallo, Irene Terrenato, Edoardo Pescarmona, Simona Di Filippo, Ferdinando Marandino, Gianluigi Ferretti, Franco Di Filippo Regina Elena Cancer Institute, Rome, Italy ABSTRACT AIMS AIMS In our series of 709 BC patients we focused on the 91 cases with micrometastatic SLN concomitantly detected by OSNA and immunohistochemistry, aimed to: correlate the copy numbers of CK19 mRNA with the risk of additional positive NSLNs; assess the relationships between the molecular subtype classification and the probability of a positive ALND; identify a subgroup of patients with a micrometastatic SLN and a negligible risk of positive NSLNs in whom ALND may be avoided. Background: Axillary lymph node dissection (ALND) may not be necessary in women with breast cancer (BC) who have micrometastasis in a sentinel lymph node (SLN), owing to the low risk of non-SLN (NSLN) involvement. In our Institute we validated and adopted the molecular diagnostic tool OSNA based on the quantitative measurement of Cytokeratin 19 (CK19) mRNA. The aims of our work in a subgroup of women with micrometastatic SLN, were: 1) to correlate the copy numbers of CK19 mRNA with the risk of additional positive NSLNs; 2) to assess the relationships between the molecular subtype classification and the probability of a positive ALND; 3) to verify whether a combination of the new above mentioned parameters is able to identify a subgroup of patients with a micrometastatic SLN and a negligible risk of positive NSLNs in whom ALND may be avoided. Methods: The SLN lysates from 709 patients were analyzed by OSNA assay. We considered only patients with a micrometastatic SLN (copy numbers between 250 and 5000/μL) and the probability of having a positive ALND was calculated by the logistic regression model. This series of BC patients were divided into four main subtypes taking in account the BC classification as defined by a combination of estrogen, progesteron receptors and HER2 status. Results: OSNA positivity for micrometastasis was reported in 91/709 cases (12,8%).The number of patients with positive ALND was 20 (22%). The statistical analyses showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers (>2000) of CK19 mRNA together with HER2 subtype. Otherwise none of the luminal A patients with a positive SLN but presenting a copy number <1000, had a positive NSLNs. Conclusions: We showed that biologically-driven analyses may be able to build new models with higher performance in terms of breast cancer axillary status prediction after positive SLN biopsy for micrometastasis. The copy numbers of CK19 mRNA and the molecular subtypes are more advantageous than traditional parameters because they are not pathologist-dependent and therefore they are more reliable and reproducible. In conclusion results obtained in our series of 91 micrometastatic cases showed that: Biologically-driven analyses may be able to build new models with higher performance in terms of breast cancer axillary status prediction. The copy numbers of CK19 mRNA and the molecular subtypes are more advantageous than traditional parameters because they provided quantitative and objective results which are more reliable and reproducible. CONCLUSIONS ASCO Annual Meeting 2012 Chigago, Illinois June 1-5, 2012 METHODS # 21A (++) macrometastasis >5000 copies mRNA/µL (+) micrometastasis250 – 5000 copies mRNA/µL (-) negative/ITC<250 copies mRNA/µL The OSNA test measures the amount of CK19 mRNA (the number of copies) that is correlated with the number of CK19 positive cells and therefore the size of the metastases CharacteristicsN. of cases% Number of patients709 Median age (range)55 (26-83) Histotype Intraductal Carcinoma628.7 Invasive Ductal Carcinoma59684.1 Invasive Lobular Carcinoma4386.0 Other81.2 Grading G1567.9 G250571.2 G314820.9 Tumor size Tis628.7 T1a8011.3 T1b12217.2 T1c29341.3 T215221.5 Lymph node status N051672.8 N1m9112.8 N18411.9 N2142.0 N340.5 Breast Cancer Subtype N. of cases Biomarker distribution ERPgRHER2Ki67 Luminal A63++NegLow Luminal B7++NegHigh HER215-/+ PosLow/High Triple Negative6Neg High Total cases91 SLNsOSNA results OSNA copy number SubtypeALND results 56+280-1000LANegative 7+1300-3200LAPositive 5+300-540LBNegative 2+3000-3600LBPositive 6+630-1700HSNegative 9+2500-3200HSPositive 4+270-830TNNegative 2+1000-1700TNPositive Total 91 Probalility of a positive ALND OR95% CIP-value Subtypes LA vs LB1.160.45-3.010.755 LA vs HS2.821.33-6.000.007 LA vs TN1.560.46-5.280.476 Grading G1+G2 vs G31.660.82-3.890.162 T T1 vs T21.520.81-2.850.191 OSNA Assay + vs ++4.762.47-9.19<0.0001 Table 5. Logistic regression analysis in 709 BC patients Table 4. Detailed analysis of the 91 micrometastatic cases Table 3. Subtype distribution in 91 BC patients with a micrometastic SLN RESULTS Table 1. Clinico-pathological characteristcs of the 709 breast cancer patients Micromestastasis in SLN detected using both CK19 immunohistochemistry fig. A and OSNA assay fig. B A B BC subtypes, tumor grading, tumor size and copy number of CK19 mRNA were studied by logistic regression analysis. As summarized in this table the model indicates that the probability to find an ALND positive is significantly higher in patients with HER2 positive tumors and with SLN OSNA ++ The number of patients with positive ALND was 20 (22%). Our findings showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers (>1000) of CK19 mRNA together with HER2 subtype. Otherwise none of the luminal A patients with a positive SLN but presenting a copy number <1000, had a positive NSLNs Table 2. Pathological characteristcs of the 91 BC patients with a micrometastatic SLN Characteristics N. Of cases% Histotype Invasive Ductal Carcinoma8391.2 Invasive Lobular Carcinoma88.8 Gradig G12123.1 G24852.7 G32224.2 Tumor size T1a1415.4 T1b1112.1 T1c4043.9 T22628.6