Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following invasive ductal carcinoma (IDC) and accounts for 10-15% of all cases. Unlike the masses or lumps formed by IDCs, ILCs grow as small, dyscohesive cells in a single-file pattern within dense layers of extracellular matrix. Paradoxically, while patients with ILC display favorable prognostic and predictive factors (Estrogen Receptor [ER]+, Progesterone Receptor+, HER2-, low Ki67), they present with more frequent long-term recurrences compared to those with IDC, indicative of endocrine resistance. Thus, there is urgent need to investigate genetic drivers of ILC disease progression in order to develop more effective therapeutic strategies and improve patient outcome. To this end, we used the Nanostring platform to measure the expression of 577 copy number variation-associated genes in 131 primary ILC tumors with long-term clinical data. This analysis identified CTTN (Cortactin; cortical actin binding protein) as a candidate ILC driver that exhibited higher expression in tumors from patients with subsequent recurrent (n=33) versus non-current disease (n=98). We further validated high CTTN mRNA/protein expression in human ILC cell lines, tumors and patient-derived xenografts, and CTTN locus (11q13.3) amplification in clinical ILC metastases to the brain, bone and ovaries. In follow-upin vitro studies, RNAi-mediated inhibition of CTTN diminished the adhesion and haptotaxis of human ILC cell lines to Collagen I, as well as impairing their growth in 3D Collagen I culture. To assess the functional role of CTTN in ILC tumor growth and metastasis, we transplanted control and CTTN knockdown human ILC cell lines into the mammary fat pads and ducts of immuno-compromised mice and monitored disease progression via bioluminescence imaging. While CTTN inhibition did not lead to a substantial impact on measurable tumor burden, both CTTN shRNAs resulted in a significant survival benefit in the fat pad model. Ongoing work is aimed at pinpointing the underlying mechanisms of CTTN's role in mediating growth in 3D Collagen I culture in vitro and disease progression in vivo. Collectively, the results of these studies will advance our understanding of ILC disease mechanisms and serve as a pre-clinical basis for improving the clinical outcome of patients with this understudied subtype of breast cancer. Citation Format: Tasdemir N, Sikora MJ, Zhu L, Levine KM, Scott J, Basudan A, Sflomos G, Sreekumar S, Bossart EA, Elishaev E, Chandran UR, Tseng GC, Jankowitz RC, Dabbs DJ, McAuliffe PF, Brisken C, Davidson NE, Oesterreich S. Investigating cortactin as a genetic driver of disease progression in invasive lobular carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-03.

Abstract Background Breast cancer recurrence is a major clinical problem for estrogen receptor positive (ER+) disease, even decades after initial surgery. These long-term recurrences are a challenge for invasive ductal carcinoma (IDC), and are particularly frequent for the histological subtype of invasive lobular carcinoma (ILC). To study the long-term endocrine resistance seen in ILC patients, our lab recently generated six long-term estrogen deprivation (LTED) models of ILC cells and performed RNA-Sequencing to identify differentially expressed genes that ostensibly allow these cells to grow in the absence of estrogen. We overlapped these results with a previously published microarray dataset of tamoxifen-resistant cells, and found that FGFR4 is the most consistently overexpressed gene in the setting of acquired resistance to endocrine therapy in ILC cells. From a recent publication of RNA-Seq from other LTED models, FGFR4 RNA overexpression is also seen in all five IDC cell lines. Hypothesis FGFR4 is an important mediator of acquired endocrine resistance in breast cancer. Methods To study the role of FGFR4 in vitro, we used multiple shRNAs and specific small molecule inhibition for growth assays. To study the role of FGFR4 in de novo resistance to endocrine therapy, we collected 129 well curated ER+ ILC tumor specimens and performed gene expression analysis on the pre-treatment samples using a custom NanoString panel. To study the role of FGFR4 in acquired resistance, we collected over 50 pairs of primary-metastatic ER+ tumors and performed exon capture based RNA-Sequencing. Results FGFR4 inhibition decreases parental and LTED cell growth in classic 2D conditions and in colony formation assays. The LTED cells, with higher FGFR4 expression, are more sensitive to its inhibition. For the parental cells, combination FGFR4 and ER-targeting drugs results in synergistic decreases in growth. In our database of primary ILC clinical samples, increased expression of FGFR4 is predictive of shorter time to distant recurrence. Among primary-recurrent tumor pairs, FGFR4 is an outlier expression gain in 20/50 (40%), spanning all recurrence sites studied (i.e. local recurrences, and metastases to the brain, bone, ovaries, and GI tract). Finally, in analyzing large cohorts of metastatic tumors, there is a significant enrichment of hotspot FGFR4 mutations in tumors originating in the breast, with >2% of metastatic ILC tumors containing such a mutation. Conclusion/Future studies FGFR4 may play an important role in de novo resistance to endocrine therapy in ILC and acquired resistance in both ILC and IDC. Ongoing studies include overexpression of wild-type and FGFR4 hotspot mutations in ILC and IDC cell lines to determine growth and metastatic phenotypes. Citation Format: Levine KM, Ding K, Priedigkeit N, Sikora MJ, Tasdemir N, Zhu L, Tseng GC, Jankowitz RC, Dabbs DJ, McAuliffe PF, Lee AV, Oesterreich S. FGFR4 is a novel druggable target for recurrent ER-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-21.

Abstract Background Invasive Lobular Carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. Distinctive properties of ILC include growth patterns, metastatic behavior, and receptor status (almost universally estrogen receptor (ER) positive). Our lab recently generated six long-term estrogen deprivation (LTED) models of ILC cells and performed RNA-Sequencing to identify differentially expressed genes compared to their parental cells cultured with estrogen. We overlapped these results with a previously published microarray dataset and found that FGFR4 is the most consistently overexpressed gene in the setting of acquired resistance to endocrine therapy in ILC cells. Hypothesis FGFR4 is an important mediator of resistance to endocrine therapy in ILC. Methods To study the role of FGFR4 in vitro, we used multiple shRNAs and specific small molecule inhibition for growth assays of ILC cells. To study the role of FGFR4 in de novo resistance to endocrine therapy, we collected 129 well curated ER+ ILC tumor specimens and performed gene expression analysis on the pre-treatment samples using a custom NanoString panel. To study the role of FGFR4 in acquired resistance, we collected over 50 pairs of primary-metastatic ER+ tumors and performed exon capture based RNA-Sequencing. Results FGFR4 inhibition decreases parental and LTED ILC cell growth in classic 2D conditions, in the setting of ultra-low attachment, and in colony formation assays. The LTED cells, with higher FGFR4 expression, are more sensitive to its inhibition. For the parental cells, combination FGFR4 and ER-targeting drugs results in synergistic decreases in growth. In our database of primary ILC clinical samples, increased expression of FGFR4 is predictive of shorter time to distant recurrence. For our collection of 50 paired, primary-metastatic ER+ tissues, FGFR4 expression increases on average >2.5 fold in the metastatic setting, with large gains even in ductal carcinoma cases. Finally, in analyzing recently published cohorts of metastatic tumors, there is a significant enrichment of hotspot FGFR4 mutations in tumors originating in the breast, with >2% of metastatic ILC tumors containing such a mutation. Conclusion FGFR4 may play an important role in both acquired and de novo resistance to endocrine therapy in ILC. Citation Format: Levine KM, Chen J, Sikora MJ, Tasdemir N, Priedigkeit N, Tseng GC, Puhalla SL, Jankowitz RC, Dabbs DJ, McAuliffe PF, Lee AV, Oesterreich S. Combination FGFR4 and ER-targeted therapy for invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-09.

Abstract Background: Invasive Lobular Breast Carcinoma (ILC) is the second most common histologic subtype of breast cancer, comprising 10-15% of all cases. ILC is clinically and molecularly distinguished from the major subtype–Invasive Ductal Carcinoma (IDC)–by loss of E-cadherin (CDH1). Studies on ILC remain sparse, in part due to limited cell culture models. The Women's Cancer Research Center (WCRC) has therefore set up a combined effort of breast surgical oncologists, medical oncologists, pathologists, and cancer biologists to collect fresh tissues and establish additional ILC cell lines. Methods: Tumor cells were grown using irradiated fibroblast conditioned media (CM) in hypoxic (5% O2) conditions. Sanger sequencing and Droplet Digital PCR (ddPCR) were utilized to test for mutation in CDH1 in tumor and circulating DNA (cfDNA), using germline DNA as control. DNA copy number status in the tumor was detected using nanoString approach. Expression of E-cadherin and a series of lineage markers was elucidated using Immunoblotting (IB) and Immunofluorescence (IF). A panel of ILC (MDA-MB-134, Sum44PE, IPH-926) and IDC (MCF-7, MDA-MB-231) cell lines was included for comparison. After establishment, WCRC-25 population doubling was compared between growth in CM or Dulbecco's Modified Eagle's Medium (DMEM), and hypoxic (5% O2) or normoxic (21% O2) conditions. Growth phenotypes were characterized in 2D, Ultra Low Attachment (ULA), and soft agar. Results: WCRC-25 was successfully established from the pleural effusion of a 77-year old patient with metastatic ILC. The patient had stage IV (T3N3M1) ER+/PR-/HER2- ILC; was treated with bilateral mastectomy, radiation therapy, and multiple lines of chemotherapy (FOLFOX due to initial misdiagnosis; 2 cycles carboplatin/paclitaxel; anastrazole, fulvestrant, pegylated liposomal doxorubicin, gemcitabine, exemastane, and eribulin (with denosumab for bone lesions); had metastatic lesions in stomach, bone, pleura, and pericardium; and ultimately passed away from progressive pleural effusions roughly 3 years after diagnosis. A novel nonsense mutation of CDH1 was observed in exon 13 (Q705*) in cell line DNA, resulting in a premature stop codon. The same mutation was confirmed in cfDNA obtained from longitudinal blood samples from the patient. Copy number analysis revealed deletion of the second CDH1 copy in tumor, and E-cadherin protein loss was confirmed by IB and IF. WCRC-25 cells expressed epithelial cell markers CK8/18 and EpCAM, and as expected did not express stromal marker αSMA. ERα expression was very low, and not sufficient for measurable hormone response. Population doubling was significantly faster in hypoxic compared to normoxic conditions, regardless of media type, but minimal in 3D. Conclusions: WCRC-25 is a novel ILC cell line, defined by CDH1 nonsense truncating mutation and LOH, resulting in loss of E-cadherin protein expression. Cells show favorable growth characteristics in hypoxic conditions and maintain epithelial-dominated protein expression. We are currently performing RNA seq analysis of the matched primary tumor and metastatic samples from the stomach, peritoneum/falciform ligament, pleural effusion and skin, which we will compare and contrast with that of the established WCRC-25 cell line. Citation Format: Kota K, Bossart E, Basudan A, Minteer T, Meier C, Brown D, Gurda GT, Miller L, Dabbs DJ, Lee A, Puhalla S, Jankowitz R, McAuliffe P, Lucas P, Oesterreich S. Generation and characterization of a novel invasive lobular breast carcinoma cell line WCRC-25 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-06-03.

Abstract Introduction The majority of publications regarding breast cancer GEPTs rarely supply detailed breast tumor histopathology in their outcome studies. As a result, the cost effective role of clinical risk assessment with histopathology of breast carcinomas tends to be minimized. The aims of this study are to characterize the details of breast tumor histopathology of patients with Oncotype Dx Recurrence Scores (RS) of 10 or less, and determine if Oncotype Dx offers value and clinical utility for patients with these low grade tumors Methods A total of 459 patients (18%) with Oncotype Dx RS of 10 or less were retrieved from a registry of 2558 patients with Oncotype Dx results. Patients had five years of follow-up with tumor registry and were treated with endocrine therapy alone. Tissue slides were available to review on 441/459 patients. Recorded details included (1) histopathologic type of carcinoma (2) mitotic score (MS), tubule formation, nuclear pleomorphism and Notttingham histologc (NG) grade. (3) Estrogen (ER) and progesterone (PgR) semiquantitated by Allred Score and Histologic Score (H Score: strong 200-300, moderate 100-199, weak <100). (4) Lymph node status. (5) overall survival and breast cancer specific survival. Results Patient ages were 33-92, with mean/median age of 60, and all had endocrine therapy alone. 148 of 441(34%) patients had carcinomas of “special types”, notable for low grade/good prognosis including tubular 22(15%), cribriform 15 (10.1%), papillary 17 (11.5%), and mucinous 28 (21%), along with 63 (42.5%) low grade classic lobular carcinomas and 3 (2%) low grade mixed ductal and lobular carcinomas. All 148 tumors had a MS of 1, were NG1 and had high ER HScores (280 median/263 mean) (Allred Scores 7-8) and high PR HScores (210 median/201 mean) (Allred Scores 6-8). The remaining 293 tumors were ductal carcinomas of no special type (NST), and 261/293 (89%) of these had a MS of 1/NG2. Of the remaining cases, 10 (3%) had a MS of 2/NG2, 18 (6%) had MS of 2/NG3 and four (1%) were MS3/NG3. Estrogen receptor H Score/Allred Score was strong (Allred Score 7-8) in 395/441 (89.6%), moderate in 45 (10.2%) and weak in 1 patient (0.2%). Progesterone HScores were strong in (Allred Score 6-8) 269/441 (76%) and moderate in the remainder. Strong and moderate ER comprised 99.8% of tumors. Thus, tumors with MS1, and NG1, all with ER HScore >200 (Allred Score of 7-8) were enriched in the RS <10, and these features distinguished this group from other tumors with a MS1. At 5 years, 433 patients (98%) were alive, 8 were dead, 1 from breast cancer due to distant recurrence. The 5-year breast cancer specific survival for this group was 99.7%. [95%CI 98.5-99.9.] 87 cases were accrued in the ongoing prospective study to date. There were 15/87 (17%) cases, 95% of which were correctly identified by pathologists as having an RS <10 using the criteria defined, with sensitivity 95%, specificity 86%, PPV 63% 95% CI(49.76-75.08), NPV 99% 95% CI(90.7-99.78). No patient had a recurrence score >22. Conclusions Pathologists can identify these low risk tumors with high accuracy. Oncotype Dx lacks clinical value and utility in this setting. Citation Format: Dabbs DJ, Serdy K, Onisko A, Clark BZ, Bhargava R, Smalley S, Perkins S, Brufsky AM. The clinical utility of oncotype Dx for patients with recurrence scores of 10 or less: A value based pathology study of tumor histopathology and outcomes analysis in an integrated delivery and finance health system [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-08-04.

Abstract Background: Invasive lobular carcinoma (ILC) accounts for 10-15% of invasive breast cancers diagnosed annually. There is increasing evidence that endocrine treatment response might differ between Invasive Ductal Carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival when other prognostic factors are taken into account. One such factor is ER status, which is more likely to be positive in ILC (90-95%) compared to IDC (60-70%). There are few studies that have directly compared mRNA and protein levels between ER+ ILC and ER+ IDC. Hypothesis: Differences in ER protein steady state levels, and/or turn-over rates contribute to differences in endocrine treatment response between patients with ILC vs IDC. Methods: We utilized publicly available TCGA data to compare ER mRNA and protein levels between ER+ ILC (n=184) and IDC (n=534). Correlation analysis with Spearman's rank order coefficient (ρ) was used to study the relationship between mRNA and protein levels. METABRIC data were analyzed to compare ER mRNA levels between ER+ ILC (n=130) and IDC (n=1152). ER H-scores and mRNA levels were also analyzed from patients with ER+ ILC (n=180) and IDC (n=1183) seen at our local UPMC Magee Womens Hospital. Finally, ER mRNA and total protein levels, and RNA and protein turn-over rates were determined in 2 IDC and 2 ILC breast cancer cell lines, using qRT-PCR and immunoblots analysis. Results: Analysis of ESR1 gene expression in the TCGA database revealed significantly lower levels of ER mRNA (Mann-Whitney, p<.0005) in ER+ ILC compared to IDC, whereas ER protein levels were similar in the two histological subtypes. The correlation between ER mRNA and protein levels is weaker in ER+ ILC (ρ=0.60) compared to ER+ IDC (ρ=0.69) tumors, though not statistically significant. The weaker correlation between mRNA and protein expression in ILC is more clear when analyzing all 130 RNA and protein pairs with available RRPA data, (ILC median ρ=0.28; IDC median ρ=0.34, p<.0005). In the METABRIC dataset, ESR1 mRNA levels were also found to be lower in ER+ ILC tumor samples compared to IDCs (Mann-Whitney, p<0.005). In concordance with these observations, the study of patients seen at our local hospital showed similar ER IHC H-scores for ER+ ILCs (H-score = 244) and IDCs (H Score = 248), despite there being significantly lower ESR1 mRNA in ILC (p<0.005). Finally, our in vitro data showed that rate of estrogen-mediated turn-over of ER protein was significantly lower in the ILC cell lines compared to the IDC cell lines, which might explain the lack of lower ER protein levels despite lower ER mRNA levels. We are currently confirming these findings in additional cell lines, and deciphering the mechanisms through the study of ER ubiquitin-modification and proteasome machinery comparing ILC and IDC. Conclusion: We have provided functional and in silico data that collectively suggest altered ER protein turn-over in ILC compared to IDC. We are currently testing if and how this affects sensitivity of ILC cells to SERDs, and underlying mechanisms. Citation Format: Sreekumar S, Levine K, Sikora MJ, Boone D, Dabbs DJ, Lee AV, Jankowitz RC, Oesterreich S. Differential turnover of estrogen receptor alpha in invasive lobular carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-02.

Abstract Background: Patients achieving pathologic complete response to neoadjuvant chemotherapy have excellent disease free and overall survival. For patients with residual disease, the residual disease in breast and lymph node (RDBN) method provides useful prognostic information. RDBN is calculated as follows: 0.2*tumor size (in cm)+lymph node status (0-3) + tumor grade (1-3). pCR, low, intermediate and high risk of recurrence categories correspond to RDBN index of 0, 0.1 to 2.9, 3 to <4.4, and ≥ 4.4, respectively. We hypothesized that the prognostic accuracy of RDBN may be improved by also taking into account the residual tumor cellularity. Methods: Retrospective review of 614 consecutive patients who underwent neoadjuvant therapy for breast cancer was performed. At our institution, tumor size/volume reduction in the breast is determined using the equation: Estimated % tumor size reduction = [(pre-therapy clinical size – “revised” pathology tumor size)/pre-therapy clinical size]*100. “Revised” pathology tumor size is calculated by multiplying the largest dimension of the gross tumor bed by the invasive tumor cellularity of the tumor bed (in comparison to the pre-therapy core biopsy sample). For example, if a 3 cm tumor bed has only 50% cellularity for invasive cancer (in comparison to pre-therapy core biopsy), the revised tumor size is 1.5 cm. Hence, we were able to use the “revised tumor size” for calculating the modified RDBN index (mRDBN). We also used gross tumor bed size for gross RDBN (gRDBN) to compare with mRDBN. mRDBN and gRDBN could be calculated on 459 of the 514 cases. Chi-Square statistical analysis was performed. Results: Mean follow up was 33.1 months (median 31, range 4-70). The results are shown in Table 1 & 2. Table 1. Overall Recurrence and MortalityRDBN Score Category Overall Recurrence Mortality nn (%)RR95% CI; pn (%)RR95% CI; pmRDBN (n=459)High5829 (50.0)19.63[7.22, 53.40]; p=<0.000118 (31.0)16.24[4.97, 53.10]; p=<0.0001Intermed16433 (20.1)7.9[2.86, 21.78]; p=<0.000115 (9.1)4.79[1.41, 16.21]; p=0.006Low803 (3.8)1.47[0.34, 6.42]; p=0.694 (5.0)2.62[0.60, 11.41]; p=0.23pCR1574 (2.5)REF 3 (1.9)REF gRDBN (n=459)High8131 (38.3)15.02[5.49, 41.09]; p=<0.000119 (23.5)12.28[3.74, 40.26]; p=<0.0001Intermed14932 (21.5)8.43[3.05, 23.26]; p=<0.000116 (10.7)5.62[1.67, 18.89]; p=0.003Low722 (2.8)1.09[0.20, 5.82]; p=12 (2.8)1.45[0.25, 8.51]; p=1pCR1574 (2.5)REF 3 (1.9)REF Table 2; Reclassification of gRDBN categoriesgRDBN mRDBN ReclassificationClassificationnLow (%)Intermed (%)High (%)Low7272 (100)0 (0)0 (0)Intermed1498 (5.4)140 (93.9)1 (0.7)High810 (0)24 (29.6)57 (70.4) Conclusions: Both mRDBN and gRDBN provide prognostic information; however, separation of categories is improved with mRDBN (Table 1). mRDBN reclassified 30% of the high risk-gRDBN patients into intermediate risk category with a recurrence rate of 20%, leaving the 'true' high risk subgroup with a revised recurrence rate of 50% (Table 2). RDBN index also identified a group of low risk patients who have prognosis similar to patients with pCR. Citation Format: Farrugia DJ, Landmann A, McAuliffe PF, Diego EJ, Johnson R, Bonaventura M, Soran A, Dabbs DJ, Clark B, Lembersky BC, Puhalla SL, Brufsky A, Jankowitz R, Davidson NE, Ahrendt GM, Bhargava R. Prognostic significance of a modified residual disease in breast and nodes (mRDBN) algorithm after neoadjuvant therapy for breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-14.

Abstract Introduction Prognosis for breast cancer patients may be determined using clinical-pathologic data (CPD) or gene expression profiling (GEP). We have previously reported a combined morphologic and immunohistochemical (IHC) method (Magee Equations) that can be used to estimate the Oncotype DX (ODX) recurrence score in clinical practice (http://path.upmc.edu/onlineTools/mageeequations.html ). MammaPrint (MP) is a 70 gene GEP assay that is used to assess prognosis. Our goal was to develop similar equations to estimate the MP result based on CPD. Methods The study included 344 patients who had MP and compete CPD in an IRB approved research setting. Using the available CPD, a logistic regression model was constructed to predict MP risk group classification. Odds ratios and corresponding 95% confidence intervals were calculated. Model estimates were used to create an equation to predict MP risk category. The risk group cut-off for predicted scores was chosen to give 0.90 sensitivity with resulting specificity of 0.51 based on the observed MP scores. This corresponds to 10% of subjects in the observed MP high risk group being predicted as low risk. The sensitivity was chosen to minimize the misclassification rate of the high risk group. Internal validation of the prediction model was determined using the bootstrap method. For each of the 1,000 iterations, the bootstrap sample was used to generate the prediction equation and the cutoff value as described above. Subsequently, this equation was used to predict the risk group for the observations not contained in the bootstrap sample (out-of-bag observations) and the resulting sensitivity and specificity were recorded. Results The logistic regression model from the observed data is: y = 0.7495(Age< 50) - 1.4135(Nottingham Grade 1) + 0.4644 (Nottingham Grade 2) + 1.7437 (Nottingham Grade 3) -0.0038*(ER H-Score)– 0.0044* (PR H-score) + 1.9220 (HER2 Positive) The probability of being classified as high risk is then given by: p=exp⁡(y)/(1+exp⁡(y)) The corresponding predicted risk group is defined as: Low Risk: p < 0.18 High Risk: p≥0.18 The 0.18 cut-off was chosen to yield 90% sensitivity. For subjects with a known MP result of high risk, the probability that the model will predict high risk is 0.90. As a result of this sensitivity, the specificity is 51%. For subjects with a known MP result of low risk, the probability that the model will predict low risk is 0.51. The sensitivity was chosen to minimize the misclassification rate of the high risk group. The median with 1st and 3rd quartiles for the cut-off value from the bootstrap prediction equations is 0.18 (0.16, 0.20). The median with 1st and 3rd quartiles for the sensitivity and specificity for the predicted scores of out-of-bag observations are 0.89 (0.85, 0.93) and 0.50 (0.45, 0.54), respectively. Conclusion The pathology data can predict for high risk MP result with a high sensitivity and moderate specificity. Citation Format: Dabbs DJ, Cooper KL, Brufsky A, Rosenzweig M, Bhargava R. Pathology data predicts MammaPrint result- The Magee MammaPrint equation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-04.

Introduction Biomarkers (BM) are often performed on core biopsies(CB) of breast tumors for treatment purposes, yet comprehensive information on BM heterogeneity is lacking. This quality assurance study is to survey BM intra-tumor heterogeneity (ITH) by comparing BM from CB and the examination of entire breast tumors from surgical excisions (SE). This interim report supplies data on 45/100 collected cases to date. Methods: Whole tumor cases, 3.0cm or less were collected in years 2013-2014, excluding neoadjuvant cases and were examined with CB by two pathologists. All tissues were processed using 2013 ASCO/CAP guidelines. Immunohistochemistry (IHC) for ER/PR/Her2/Ki67 was performed on breast core biopsies and each block of a tumor surgical excision (1-13 blocks per case). IHC was performed using the Ultra Ventana-Roche device with FDA IVD clones ER (SP1), PR(1E2) Her2(4B5) Ki67(30-9). Whole slides were semi-quantitated for ER/PR using HScore (0-300), Her2 was interpreted with 2013 ASCO/CAP guidelines and Ki67 proliferation index (PI) as estimated whole slide percent of tumor cell nuclear stain. Mean HScores and PI were calculated for all BM on SE, and CB deviations from the SE mean were calculated for ER/PR/Ki67. ITH of BM was assessed by comparing results among all tissue blocks for each case. Her2 results were descriptive. Eight cases lacked CB Ki67 PI. Results There were no cases in which categorical hormone receptors results (positive/negative) differed between CB and SE. 2/45 (4%) cases were Her2 positive, and 13/45(28%) were equivocal by IHC with one case amplified by in situ hybridization. Comparing ER between CB/SE , 3/45 differed in HScores by 30% or more. 10/45 SE ER had ITH of 25% or more among different tissue blocks. 14/45 cases had CB/SE PR difference in HScore of 30% or more. 18/45 cases had SE PR with ITH HScore of 25% or more between tissue blocks. Ki67 PI differed between CB/SE by more than 25% in 18/37 cases, with ITH among tissue blocks of more than 25% in 23/37 cases. The variability of CB-SE results and ITH among tissue blocks are summarized in table 1. Conclusions In this interim report of 45/100 cases, (1) ITH of 25% or more occurred for ER content in 22 % of cases (2) similar ITH exists for PR content in 40% of cases. (3) Ki67 PI tissue block ITH was expressed with variation of 25% or more in 23/37 cases. (4) While this degree of variability of BM may not affect treatment decisions, the tissues show sufficient BM variability that should stimulate concern for accuracy of results of prognostic gene expression profiles and genomic testing of breast tumors. Citation Format: Dabbs DJ, Clark B. Breast cancer biomarkers: Global tumor biomarker quality assurance study of intratumor heterogeneity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-15.

Abstract Background: Patients with invasive lobular carcinoma (ILC) would be expected to have favorable outcomes compared to patients with invasive ductal carcinoma (IDC) given that ILC is more often hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative, of lower grade, and displays decreased proliferation markers. Based on our preclinical studies showing differential hormone response in HR+ ILC vs. IDC and on recent studies suggesting differences in endocrine treatment response between patients with ILC vs. IDC, we designed a biomarker-driven, neoadjuvant window trial for newly diagnosed women with HR+, HER2-negative ILC. We hypothesize that Ki67 will be reduced by 85% in the fulvestrant arm compared with 60% and 75% reduction in the tamoxifen and anastrozole arms, respectively, and that Ki67 reduction will correlate with alterations in expression of ER and ER-regulated genes. Differential Ki67 effect will serve as a surrogate for outcome of patients with ILC on endocrine therapy. Trial Design: This multicenter study (NCT02206984) will enroll 150 women with HR+ and HER2-negative ILC. A mandatory research breast tumor biopsy will be performed at baseline. Fifty patients will be randomized to each of three open-label treatment arms for 21 days: fulvestrant (two 250 mg IM injections on both day 1 and day 14), anastrozole (1mg orally daily), or tamoxifen (20 mg orally daily). Biomarkers of response will be assessed on baseline and post-treatment tumor tissue. Patients will proceed to definitive surgery on day 21 after study drug exposure, or they will undergo a second research breast core biopsy if further neoadjuvant treatment is planned. Eligibility Criteria: Eligible patients include postmenopausal women with newly diagnosed, HR+, HER2-negative ILC (excluding pleomorphic subtype) measuring ≥ 1cm, with adequate organ function, ECOG PS ≥ 2, and agreeable to baseline research breast tumor biopsy. Specific Aims: The primary endpoint is percent change from baseline to post-treatment Ki67 values in ILC tissue after 21 days of endocrine treatment. Comparisons across study arms will be made using a general linear model adjusting for institutional effect, with 80% power estimated for pairwise comparisons of log2(% staining) between treatment arms, allowing for 10% attrition. Secondary endpoints include post-therapy Ki67, and change in ER and PR protein expression by IHC. Finally, planned correlative studies include evaluation of gene expression, epigenetic markers, and DNA sequence variants in ILC tissues in an effort to identify biomarkers of endocrine response and putative drivers of endocrine resistance in ILC. Target Accrual: This study will be open to enrollment by August 2015 at the University of Pittsburgh. Additional sites will be opened through the Translational Breast Cancer Research Consortium (TBCRC). We anticipate an accrual rate of 8 patients per month. (Funding from Susan G. Komen® and AstraZeneca). Citation Format: Jankowitz RC, McAuliffe PF, Sikora MJ, Butler L, Ahrendt G, Johnson R, Diego E, Bonaventura M, Puhalla S, Lembersky B, Clark B, Brufsky A, Kurland BF, Davidson NE, Dabbs DJ, Oesterreich S. A neoadjuvant window trial of endocrine response in women with invasive lobular carcinoma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-14.

Abstract BACKGROUND: Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive aggressive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. MATERIALS AND METHODS: From 2/2014 to 4/2015, patients with percutaneous core needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Under supervision of the surgical pathologist, fresh tissue measuring between 5-15 mm in length was taken from lumpectomy or mastectomy specimens. Tissue was divided such that half was mechanically and enzymatically dissociated and then cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and the second half, known as the "mirror image" remained as part of the clinical specimen. RESULTS: Of 49 consented patients, mean age was 59 ± 10 years. 7 were excluded due to final pathology not consistent with DCIS: 4 upstaged to invasive ductal cancer, 2 had microinvasion and 1 showed pleomorphic lobular carcinoma in situ. Of the remaining 42, 9 were failures: 5 tissues were not received in lab and 4 cases were received, but no cells grew in culture. Of the remaining 33 cases of DCIS, 70% (n=23) and 27% (n=9) were nuclear grade 2 and 3 respectively. 91% (n=30) were ER-positive, with H-score ranging between 4 and 300. 19 (58%) were expanded in cell culture for up to two months in culture, and 14 were frozen immediately after mechanical dissociation for future growth. The 19 cell cultures could be cryopreserved and expanded. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal cells and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and when conditioned media and ROCK inhibitor were withdrawn. When grown to 100% confluency, the cultures appear to organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. CONCLUSION: Primary cultures of DCIS derived directly from patient tissues may serve as in vitro models for the study of DCIS. Citation Format: McAuliffe PF, Brown DD, Oesterreich S, Lee AV, Johnson RR, McGuire KP, Davidson NE, Brufsky AM, Dabbs DJ. Developing in vitro models of ductal carcinoma in situ from primary tissue. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-02.

There are a few studies that have evaluated a panel of stains on a single large data set of breast cancers, which is required for direct comparison between antibodies. The immunohistochemical panel in this study was chosen to include breast-specific markers and markers that are expressed in tumors resembling breast cancer. The individual marker positivity in decreasing order was 95% (177/186) for GATA-3, 92% (172/186) for cytokeratin (CK)7, 80% (151/189) for AR, 80% for estrogen receptor (158/198), 69% for progesterone receptor (137/198), 55% (105/190) for NY-BR-1, 52% (99/189) for mammaglobin, 31% (59/191) for vimentin, 26% (51/195) for GCDFP-15, 0.5% (1/186) for CK20, and 0% (0/188) for PAX-8. When tumors were categorized based on estrogen receptor and HER2 status; a total of 45 profiles were identified. In addition, some tumors showed an unconventional profile-although the majority of breast carcinomas were CK7-positive/CK20-negative, a CK7-negative/CK20-negative profile was seen in ∼8% of the cases. Such a profile can create confusion in investigation of a carcinoma of unknown origin. The results define the individual sensitivity of each marker and establish a baseline diagnostic profile of breast cancer in a large data set. In addition, the results support the use of immunohistochemical panel for confirming or determining breast as the source of metastasis.