McCullough, AE

Abstract Introduction: Striking clinical responses to immunotherapies in subsets of patients with a variety of solid tumors have prompted the search for predictive biomarkers. Recent studies have proposed that gene specific and genome wide mutation and copy number signatures in tumor cells may be predictive of responses to immune checkpoint blockade. Notably high levels of PD-L1 on tumor cells, a context associated with an adaptive immune response, has been linked to specific oncogenic driver lesions including loss of PTEN, activating KRAS mutation, and MYC amplification, and to the total burden of copy number variants (CNVs) in aneuploid tumors. Triple negative breast cancers (TNBCs) typically have multiple driver mutations and high levels of CNVs in their genomes. Thus there is significant interest in exploiting genomic data for the development of prognostic immunotherapy biomarkers for patients with TNBC. Study Design: We interrogated 62 well annotated surgical resections from patients with TNBC and assessed the associations of genomic lesions with expression of PD-1 and PDL-1 in tumor and non-tumor cells in each sample. We applied a systematic approach to rigorously interrogate the genomes of each TNBC sample. Tumor ploidy was initially measured with DNA content flow cytometry followed by sorting the nuclei of distinct diploid, tetraploid, and aneuploid cell populations from each TNBC. The next level of analysis measured genome wide copy number variants (CNV) with oligonucleotide arrays designed for CNV detection using purified (>95%) tumor populations. This enabled the discrimination and mapping of CNVs including single copy losses and gains, focal amplifications, and homozygous deletions across each TNBC genome. Finally we generated whole exome data in a subset of samples to increase the resolution within loci of interest and to incorporate mutations of individual genes into our genomic signatures. This combined approach provides high resolution measures of TNBC genomes from ploidy, whole chromosome and chromosome arm level CNVs, focal amplicons, breakpoints and homozygous deletions, to the level of gene specific indels and mutations. In parallel whole tissue samples were screened by IHC for PD-1 and PD-L1 using validated antibodies and established scoring methods for staining of tumor and non-tumor cells. Results and Conclusions: High levels of PD-1 and PD-L1 were detected in 5/60 (8.3%) and 16/60 (26.7%) evaluable cases with staining of PD-1 exclusively on non tumor cells, while PD-L1 was primarily on tumor cells; 15/16 (93.8%) cases. These data were then used to test the associations of individual recurring genomic lesions, and the extent and nature of chromosome aberrations on the expression patterns of PD-1 and PD-L1. Homozygous deletion of PTEN or activating mutation in PIK3CA did not correlate with increased expression of either immune checkpoint regulator in TNBC cells. Furthermore tumors with highly aberrant aneuploid genomes and distinct oncogenic drivers frequently express relatively low levels of both proteins suggesting an intrinsic escape from immunosurveillance. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Yearley JH, Annamalai L, McCullough AE, Anderson KS, Pockaj BA. Genomic lesions and PD-1/PD-L1 expression in resected triple negative breast cancers [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 P2-07-05.

Abstract Introduction: The programmed cell death-1 (PD-1) immune checkpoint is a normal regulator of the autoimmune response to inflammatory stimuli. Several cancers have taken advantage of this mechanism by harboring its ligand (PD-L1), thus activating the checkpoint to evade immune destruction. Increased expression of PD-L1 on tumor cells has been linked with worse outcomes in several cancers, including breast cancer. In triple-negative breast cancer (TNBC), expression of PD-L1 is increased compared to luminal cancers and as such, has become a favorable target for anti-tumor therapies. However, few studies evaluate the impact of PD-1 and PD-L1 expression on overall survival (OS) in TNBC. The aim of this study was to assess OS based on PD-1 and PD-L1 positivity and distribution in the tumor microenvironment. Methods: All patients with newly diagnosed TNBC from 1999 to 2015 with adequate pathologic specimen were identified for evaluation. Surgical pathology specimens were stained by immunohistochemistry (IHC) for PD-1 and PD-L1. Those considered PD-1+ or PD-L1+ had a staining score of 3, 4, or 5. Patient and clinical characteristics of the adjuvant group were compared by PD-1 and PD-L1 status with Chi-Square and ANOVA, as appropriate; characteristics of the neoadjuvant population were described. OS was calculated using Kaplan-Meier method and compared by Log Rank test for PD-1, PD-L1 positivity and tumor microenvironment status. Results: Fifty-two patients met inclusion; 38 had surgery prior to chemotherapy while 14 had neoadjuvant chemotherapy. There were 15 PD-1+ patients (39%) and 21 PD-L1+ (55%) patients in the treatment naive cohort. No significant difference was found in the treatment naive group between age, race, postmenopausal status, final stage, or pathologic characteristics (e.g. tumor size, mitotic rate, and grade) for all PD-1+ and PD-L1+ patients. Of the 21 PD-L1+ patients, staining was present in the tumor, tumor/non-tumor (mixed), and non-tumor microenvironment 42.9%, 33.3%, and 23.8% of the time, respectively (p=0.008). No significant difference was found in OS between PD-1+ and PD-1- patients (p=0.509) or PD-L1+ and PD-L1- patients (p=0.240). However, distribution of PD-L1 staining significantly associated with OS. Those demonstrating PD-L1 staining in the tumor had a significantly higher OS than those with PD-L1 staining in the non-tumor microenvironment (p=0.008). In the subset of PD-L1+ patients, those with staining in the tumor show a 10-year OS of 100% versus 60% for those with staining in non-tumor (p=0.006). In the neoadjuvant cohort, 14.3% of patients expressed PD-1 positivity, while 42.9% had PD-L1 positivity; the distribution of the PD-L1 staining in tumor 14%, mixed 29%, and non-tumor 50%. No difference in OS was found based on PD-1 or PD-L1 (p=0.660) positivity in the neoadjuvant cohort. Conclusion: A large number of TNBC stain for PD-1 and/or PD-L1. The implications of this are yet to be determined. In this small cohort, pattern of staining had a significant impact on overall survival in treatment naïve patients. This finding will need to be validated on a larger cohort but may have important implications for treatment by defining patients who may benefit by checkpoint inhibitor therapy. Citation Format: Linnaus ME, Kosiorek H, Barrett MT, Dueck A, Anderson KS, Ocal IT, McCullough AE, Annamalai L, Yearley JH, Pockaj BA. Effect of PD-1 and PD-L1 in the tumor microenvironment on overall survival of triple-negative breast cancer patients [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 PD5-03.

Abstract Introduction: Previously, we detected amplification of chromosome 9p24.1 encoding PDL1, PDL2, and JAK2 (the PDJ amplicon) in up to 25% of triple negative breast cancers (TNBC) using oligonucleotide CGH arrays (aCGH). Amplification was associated with poor outcome and activation of the JAK/STAT pathway. Here, we have developed a novel fluorescence in situ hybridization (FISH) for detection of the JAK2/PDL1 amplification. Methods: We selected five 9p24.1-amplified and five non-amplified paraffin embedded TNBC tumor samples, defined by aCGH log2ratio>2.0 as amplified for FISH validation. 5' JAK2 DNA labeled with SpectrumGreen dUTP, 3' JAK2 DNA labeled in SpectrumOrange dUTP and a commercially available chromosome 9 centromeric probe (Spectrum Aqua) were combined as one probe set. The break-apart (BAP) probe set was applied to individual slides, hybridized, and washed, 50 events/sample were counted. We defined 9p24.1 amplification by FISH as the ratio of average JAK2 score/ average centromere 9 (CEN 9) score >1.1. Nonparametric student's t-test was used for statistical analysis. Results: In the amplified subgroup (n=5), JAK2 amplification was detected by FISH with the range from 1.89 to 21.0 (mean, 6.76). To adjust for aneuploidy, the ratio of JAK2 to CEN 9 was measured with the range from 1.02 to 9.21 (mean ratio, 2.9). The sample with highest level of amplification (aCGH log2ratio =4) detected by aCGH also scored highest by FISH (FISH ratio=9.21). One case with JAK2 amplification (aCGH log2ratio =3) was not detected by FISH (ratio, 1.02), but had low tumor cell content. In the non-amplification subgroup (n=5), JAK2 amplification was detected by FISH with the range from 0.98 to 3.54 (mean, 1.8), the ratio of JAK2 to CEN 9 was measured with the range from 0.48 to1.05 (mean, 0.73). Of note, two tumors with copy number loss by aCGH were confirmed by FISH (ratio 0.48, 0.56). In total, the 9p24.1 amplification was detected in 4 out 5 (80%) amplified samples defined by aCGH and 5 out 5 not amplified. A significant difference in JAK2: CEN 9 ratio (p=0.03) was observed between the amplified and non-amplified subgroups but not in JAK2 absolute scores (p=0.095). Conclusion: In this study, we have developed a novel FISH assay for detection of the 9p24.1 amplification in TNBC, encoding JAK2, PD-L1, and PD-L2. The FISH assay correlates with detection of the amplification by aCGH, but is less sensitive, in particular in tissue with lower tumor cell content. We predict that 9p24.1 amplification will be a clinically relevant biomarker in TNBC. Citation Format: Chen M, Andreozzi M, Pockaj B, Barrett MT, Ocal IT, McCullough AE, Anderson KS. A novel fluorescence in situ hybridization assay to detect 9p24.1 amplification in triple negative 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 P6-07-17.

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous disease. Amplification of chromosome 9p24.1 encoding JAK2 and PD-L1 has been reported in up to 25% of TNBC and is associated with poor clinical outcome. In lymphoma, JAK2 is a transcriptional activator of both PD-1 ligands, and chromosome 9p copy number gain has been associated with therapeutic activity of nivolumab. We evaluated the interaction of JAK2 and PD-L1 expression in TNBC. Methods: 9p24.1 amplification in 4 TNBC cell lines (MDA-MB-231, MDA-MB-436, HCC1937, and HCC70) was measured using array comparative genomic hybridization (aCGH). Amplification was defined as aCGH log2ratio>2.0. Cell surface expression of PD-L1 was detected by flow cytometry and compared with the median fluorescence intensity (MFI) of isotype control Ig. To selectively inhibit JAK2, lentiviral vectors encoding two different shRNA were generated. JAK2, pSTAT1 and pSTAT3 expression were measured by immunoblot. The effects of the anti-JAK1/2 inhibitor ruxolitinib and interferon-gamma (IFN-γ) induction of PD-L1 was measured. Results: 9p24.1 copy number loss was measured in MDA-MB-231 (log2ratio =-1), neutral in HCC1937 ((log2ratio =0), gained in MDA-MB-436 ((log2ratio =+1) and amplified in HCC70 ((log2ratio =+2). No correlation was observed between PD-L1 expression and the 9p24.1 amplification, with the MFI ratio range from 5 to 16.5, mean 8.3 by flow cytometry. TNBC cell lines had higher baseline expression of PD-L1 compared to the ER+ cell lines T47D and MCF-7 (ratio, 0 p=0.1). Low dose IFN-γ (1.0-10.0 ng/ml) rapidly induced expression of PD-L1 in MDA-MB-231 (1.5 fold increase) and HCC70 (3.3 fold increase) with significant activation of the JAK2/STAT1 pathway. The induction of pSTAT1 and PD-L1 expression by IFN-γ was blocked with low dose (1mM) JAK1/2 inhibitor ruxolitinib. Knockdown of JAK2 with shRNA (>80%) did not impact PD-L1 baseline expression in MDA-MB-231 and HCC70 but abrogated IFN-γ –mediated induction of PD-L1 and the phosphorylation of STAT1. Conclusion: These data suggest that TNBC cell lines have baseline PD-L1 expression, but the cells with 9p24.1 amplification are highly sensitive to PD-L1 induction with IFN-γ which can be abrogated with inhibition with a JAK1/2 inhibitor or shRNA. Synergistic inhibition of PD1/PD-L1 and JAK2 may have therapeutic efficacy in the subset of TNBC with 9p24.1 amplification, and the dynamic effects of tumor PD-L1 expression in response to local inflammation should be considered in the evaluation of PD-1/PD-L1 checkpoint blockade. Citation Format: Chen M, Pockaj B, Andreozzi M, Barrett MT, Ocal IT, McCullough AE, Krishna S, Anderson KS. JAK2 and PD-L1 amplification enhance the dynamic expression of PD-L1 in triple negative 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 P2-04-18.

Abstract Introduction: Homologous recombination (HR) is essential for repairing double-stranded DNA breaks. Tumors deficient in DNA HR display high levels of copy number aberrations including amplifications, deletions, and breakpoints. Studies in BRCAmut cancers have shown that HR deficiency (HRD) renders triple negative breast carcinoma (TNBC) sensitive to carboplatin or Poly(ADP-ribose) polymerase (PARP) inhibitors via a synthetic lethal interaction, as previously described. Clinical observations in breast and other solid tumors suggest that the HRD phenotype may arise independently of BRCA1/2 mutation. The implications of HRD enrichment in PD-L1, PD-L2, Jak2 (PDJ) 9p24.1 amplified TNBC are not known. Our goal was to determine the HRD status in PDJ enriched tumors, and the potential association of the PDJ amplicon with a BRCA like HRD phenotype. Methods: Archival fresh frozen and formalin-fixed paraffin embedded (FFPE) tumor samples from 41 patients with TNBC were obtained from 1998-2015. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted using DNA content flow cytometry and interrogated with oligonucleotide array comparative genomic hybridization (aCGH). Tumor homologous recombination deficiency (HRD) scores were calculated by number of interstitial aberrations (IA) in their genomes (HRD-IA). A cut-off of < 40 (low), > 40 (high) or > 50 (very high) was assigned based on our studies of known BRCAmut tumors. Data were compared between HRD groups using chi-square, ANOVA F-test or log-rank test. Results: 14/41 (34%) TNBC patients had high HRD-IA score and 6/41 (14.6%) had very high HRD-IA score. The median age was 61.8 years (SD = 12.8), the average tumor size was 3.1cm (SD = 2.6), 42% were node positive, 24% were pre-menopausal, 19% were Black, Hispanic, or Asian/Pacific Islander. Genetic testing was negative for BRCA1/2 mutations in 9/41 patients, including 3 high and very high HRD-IA score patients. There was no difference in the OS at 5-years [74% versus 59% (p = 0.907)] in the high HRD-AI group. 12/41 (29%) patients were PDJ+. In the PDJ+ patients, 4/12 (33.3%) had HRD > 40 and 1/12 (8.3%) had HRD > 50 compared to the PDJ- patients where 10/29 (34%) had high HRD-IA score and 5/29 (17%) had a very high HRD-IA score, p=0.463. Patients with inflammatory TNBC were significantly more likely to have high HRD-IA score 4/6 (66%) compared to non-inflammatory TNBC 8/31 (26%), p=0.05. Conclusions: We describe a unique group of TNBC patients with similar patient characteristics, tumor size, nodal status, and stage when compared by low, high and very high HRD-IA score. For inflammatory TNBC, there was statistically significant percentage of patients with a high HRD-IA score. We did not see a statistically significant correlation with 9p24.1 amplification based on HRD-IA score which has been suggested in other data sets. The HRD-IA score did not statistically impact OS but there was a suggestion that those with a higher score may have an improved OS potentially due to chemotherapy sensitivity. Further study with a greater number of patients to adequately power the study is needed to determine whether our initial findings can be confirmed and whether the HRD-IA score can be used to define a specific subset of TNBC patients. Citation Format: Gawryletz CD, Anderson KS, Northfelt DW, Kosiorek HE, Linnaus ME, Ocal IT, McCullough AE, Pockaj BA, Barrett MT. Homologous recombination deficiency in PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast 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 P1-05-19.

Abstract Introduction: Clonal heterogeneity in cancer is associated with resistance to therapies and evolution of metastatic disease. Clinical management of breast cancer relies on the status of estrogen (ER), progesterone (PR), and Her2 receptors in diagnostic biopsies. Results for these biomarkers rely on IHC and FISH assays that incorporate staining intensity and percentage of positive cell numbers for each sample of interest. Heterogeneous results for one or more of these biomarkers suggest the presence of multiple tumor populations within a biopsy. However the impact of clonal heterogeneity on clinical biomarkers has not been rigorously evaluated. Methods: We interrogated the clonal composition of 3 treatment-naïve surgically excised invasive ductal carcinomas. Patient #1 cancer, (pT2, pN0) was grade 3, ER+, PR+, and Her2-- with a germ line BRCA2Q49X mutation. Patient #2 cancer (pT2, pN0) was grade 3, ER+ PR- Her2+. Patient #3 was a locally advanced cancer with both axillary and supraclavicular lymph node (LN) involvement (pT2, pN3c, and was grade 3, ER+PR-Her2+ by core but found to be ER+PR-Her2- on the resection specimen. We applied DNA content flow cytometry to multiple (8-18) mapped biopsies in each case. Each sorted tumor population was interrogated with whole exome sequencing, and whole genome array comparative genomic hybridization (aCGH). Single cell analysis of aneuploid populations present in the primary tumor and lymph nodes in patient 3 was done to further assess clonal heterogeneity. Results: Tumor from patient #1 had a single ploidy with stable copy number profiles including an interstitial 13q deletion that converted a germ line BRCA2Q49X mutation to homozygosity, and a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Tumor from patient #2 had a dominant ploidy (3.2N) in each of 10 (A1-A10) primary tumor biopsies but with a second co-existing ploidy (3.6N) in one (A2) biopsy. A homozygous somatic BRCA2r3129x mutation was identified in all 10 biopsies and in both ploidies; however the genomes had heterogeneous aCGH profiles. In contrast, 12 primary biopsies from Patient #3 contained 6 distinct ploidies with highly aberrant but homogenous genomes, characterized by SARC amplification, homozygous deletion of ROBO1 and ROBO2, and clonal mutations in TP53, NF1, and PIK3CA. One of the ploidies, (5.8N) was present in an adjacent node and another (5.0N) in both the adjacent and distant nodes. Single cell analyses of the 5.0N and 5.8N populations revealed that allele frequencies of driver mutations were stable in the tumor and become homozygous in the LNs. There were no Her2 amplicons or mutations in biopsies analyzed in Patients #2 and #3 further contradicting the heterogeneous staining for ER, PR and Her2. Conclusion: Tumor heterogeneity includes variations in ploidies, copy number aberrations, and allele frequencies. However even highly aberrant aneuploid genomes can be stable within multiple primary and lymph node sites. Rigorous interrogation of flow sorted tumor populations, in contrast to inferring heterogeneity in bulk samples, identifies driver aberrations in single biopsies, challenges heterogeneous results with clinical biomarkers, and distinguishes allelic heterozygosity from tumor heterogeneity in highly aberrant aneuploid tumor genomes. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Webster T, Wison Sayres MA, McCullough AE, Anderson KS, Pockaj BA. Clonal heterogeneity in breast cancer and its impact on clinical biomarkers [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 P1-05-12.

Abstract Introduction: We have previously identified a 9p24.1 amplicon targeting PD-L1/JAK2 (PDJ) in subset (26.7%) of triple negative breast cancer (TNBC) patients. The PDJ amplicon is common in newly diagnosed and chemotherapy treated TNBCs and is associated with a worse prognosis. Our goal in this study was to determine whether immunohistochemistry (IHC) evaluation could identify those patients who harbor the PDJ amplicon. Methods: TNBC patients from 1999 to 2015 whose tumors were flow-sorted and evaluated by array-based comparative genomic hybridization (CGH) were identified; paraffin slides were obtained for IHC staining evaluation of JAK-2, phosphorylated STAT3 (pSTAT3), and PD-L1. Pathologic analysis consisted of scoring the stains for intensity (0-3+) and the relative percent of tumor cells with positive staining; positive score was defined as 3+ for any percent staining. Statistical analysis of IHC staining was performed to determine association with the PDJ amplicon defined by focal 9p24.1 copy number gain with CGH log2ratios of >1.0 for each sorted TNBC sample. Results: Eleven of 43 TNBC patients evaluable by IHC had the PDJ amplicon. There was no association between PDJ amplification and IHC staining for JAK-2, pSTAT3, or PD-L1 regardless of staining intensity or percentage tumor cells positive. Table 1 describes PDJ amplicon status and positivity for JAK2, pSTAT3 and PD-L1. Of PDJ-positive samples, 64%, stained positive for JAK2, 27% positive for pSTAT3, but 0% for PD-L1 in tumor cells. However, in the PDJ-negative group, 69% still stained positive for JAK2, 19% positive for pSTAT3, and 9% for PD-L1. Table 1: PDJ amplicon status and IHC staining JAK2 (3+) (n;%)pSTAT3 (3+) (n;%)PD-L1 (3+) (n;%)PDJ - (n=32)22;69%6;19%3;9%PDJ + (n=11)7;64%3;27%0;0% JAK2: Most PDJ-positive samples (10/11, 91%) showed some JAK-2 staining. Although one had only 1+ staining at 5% of the cells, the majority (7/11, 64%) had staining of 3+ and >10% of the cells with 2 patients demonstrating 3+ staining of 75% and 90% of the cells. However, those patients without the PDJ amplicon also exhibited JAK2 staining, with 66% (n= 22) of PDJ-negative cases staining strongly for JAK-2 (3+) and > 10% of cells. pSTAT3: All but one PDJ-positive case demonstrated some staining (1+ or greater) for pSTAT3; the staining intensity and percent positivity were much less than the JAK-2 staining with only 2 cases (18%) having 3+ staining of > 10%. Similar staining was seen in the PDJ-negative cohort; 75% of PDJ-negative patients had some pSTAT3 staining (1+ or greater) and 1 patient (5%) exhibited 3+ staining > 10%. PD-L1: Finally, the PD-L1 staining was low overall with only 18% of PDJ-positive cases demonstrating some staining for PD-L1. Notably, only one case stained 3+ with 20% positivity while the majority of the samples had a 2+ intensity encompassing 5-50% of the cells. Conclusions: IHC staining for JAK2, pSTAT3, or PD-L1 was not associated with the presence of the PDJ amplicon. Notably positive IHC staining for JAK2 was observed for both PDJ-positive and PDJ-negative tumor cells, thereby nullifying its application as a screening tool for PDJ amplification. Alternative methods, such as fluorescence in situ hybridization, are needed to identify PDJ-positive patients for further study. Citation Format: Linnaus ME, Kosiorek H, Ocal IT, Dixon L, Barrett MT, Gawryletz CD, Anderson KS, McCullough AE, McEachron TA, Pockaj BA. Immunohistochemistry cannot be used to detect PD-L1/JAK-2 amplification [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 P1-03-13.

Abstract Background Women with benign breast disease (BBD) have an increased risk for breast cancer (BC). Almost 30% of all BCs develop in women with prior BBD. Information about the features of the expected BCs after BBD would enable individualized surveillance and prevention strategies for these women. We sought to characterize BCs developing in a large cohort of women with BBD. Methods Our cohort includes 13,485 women who underwent breast biopsy for mammographic or palpable concern between 1967-2001. Biopsy slides were reviewed and classified as nonproliferative (NP), proliferative disease without atypia (PDWA), or atypical hyperplasia (AH). BCs were identified by follow-up questionnaires, medical records, and Tumor Registry data. BC tissues were obtained and reviewed. Results With a median follow-up of 15.8 years, 1273 women developed BC. Most BCs were invasive (81%), among which 61% were ductal, 13% mixed ductal/lobular, and 14% lobular. Two thirds were intermediate or high-grade, and 29% were node positive. Cancer characteristics were similar across the three histologic categories of BBD, with similar frequency of DCIS and invasive disease, tumor size, time to invasive BC, histologic type of BC, nodal positivity and HER2 positivity. Women with AH had a higher frequency of ER+ BC (91%) vs women with PD (80%) or NP (85%), p=0.02. Conclusion A substantial proportion of all BCs develop in women with prior BBD. We show that the majority of BCs after BBD are invasive tumors of ductal type, with a substantial proportion node positive. 84% of cancers were ER-positive. Chemoprevention should be strongly encouraged in higher-risk women with BBD. Citation Format: Visscher DW, Frost MH, Hartmanan LC, Frank RD, Vierkant RA, McCullough AE, Winham SJ, Vachon C, Ghosh K, Brandt KR, Farrell AM, Tarabishy Y, Hieken TJ, Haddad TC, Kraft RA, Radisky DC, Degnim AC. Clinicopathologic features of breast cancers that develop in women with previous benign breast disease. [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 P6-10-19.

Abstract Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast 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 P6-03-13.