Pockaj, BA

Abstract Background: Circulating tumor DNA (ctDNA) analysis holds potential for minimal residual disease (MRD) detection in early stage breast cancer. However, sensitivity for MRD is limited due to low ctDNA levels in early stage patients and limited blood volumes. Loss of input DNA during library preparation, limited multiplexing or low sensitivity of current molecular methods further limit accuracy. To address this gap, we have developed TARgeted DIgital Sequencing (TARDIS), a novel method for simultaneous analysis of multiple patient-specific mutations in plasma DNA. Methods: Using tumor exome sequencing, we identify and prioritize somatic founder mutations, design nested primers and evaluate them for multiplex performance. Using 5-10 ng input plasma DNA, we perform 1) targeted linear pre-amplification to improve downstream molecular conversion, 2) single-stranded adapter ligation to incorporate unique molecular identifiers (UMIs) and 3) targeted PCR to prepare sequencing-ready libraries. The resulting sequencing reads have fixed target-specific ends and variable ligation ends. We utilize fragment size and UMIs to group sequencing reads into read families. To ensure specificity, we require targeted mutations are supported by 2 or more read families. Results: To assess analytical performance, we targeted 8 mutations in cell-free DNA reference samples with 0.25%-2% mutation allele fractions (AFs). Precision across 7-16 replicates at each AF level agreed with expectations of Poisson distribution, demonstrating effective analysis of ˜70% of input DNA. At 2%, 1%, 0.5% and 0.25% AFs, variant-level sensitivity was 96.4%, 96.4%, 91.1% and 65.8%, approaching the theoretical limit given input DNA. At 0.25% AF, 3-7 mutations were detected per sample, achieving 100% sample-level sensitivity. In 16 wild-type replicates, no targeted mutations were called (100% specificity). Averaging multiple mutations improved precision in sample-level AF estimates. Mean AFs from 8 mutations for the 2% sample were 2.34%-2.80% (5.8% CV). In 6 patients with breast cancer treated with neoadjuvant therapy (NAT), we analyzed 8-18 patient-specific mutations (mean 11.8). Before treatment, ctDNA was detected in 5/6 patients at mean AFs of 0.02%-1.19% (mean 0.40%), supported by 2-10 mutations (mean 5.6). Of these 5 patients, 4 had residual disease after NAT and ctDNA was detected pre-operatively or during NAT in 3/4 patients. 1 patient achieved pathological Complete Response and ctDNA was undetectable after NAT. Conclusions: Preliminary results suggest TARDIS enables accurate MRD detection after neoadjuvant therapy in patients with early stage breast cancer. On-going work is expanding this analysis to include additional patients and investigate the clinical validity of peri-operative ctDNA monitoring. Summary of clinical resultsPatientPre-NAT Stage (TNM)SubtypeNo. of Mutations TargetedBaseline ctDNA (AF%, No. of Mutations)ctDNA after or during NAT (AF%, No. of Mutations)Residual Tumor (TNM)1T3 N1ER+ PR+ HER2-8+ (0.02%, 2)-T2 N12T3 N0TNBC12+ (0.29%, 6)+ (0.01%, 1)T1a N03T2 N1TNBC18+ (1.19%, 10)+ (0.01%, 1)T1mi N04T3 N1TNBC10+ (0.02%, 3)+ (0.05%, 3)T3 N15T2 N0TNBC9+ (0.46%, 7)-pathCR6T1c N1TNBC14--pathCR Citation Format: McDonald BR, Contente-Cuomo T, Sammut S-J, Ernst B, Odenheimer-Bergman A, Perdigones N, Chin S-F, Farooq M, Cronin PA, Anderson KS, Kosiorek H, Northfelt D, McCullough A, Patel B, Caldas C, Pockaj B, Murtaza M. Multiplexed targeted digital sequencing of circulating tumor DNA to detect minimal residual disease in early and locally advanced breast cancer [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 P4-01-21.

Abstract Background Survivors of ER-expressing operable breast cancer (ER+BC) generally do not receive hormone replacement therapy for menopausal symptoms due to concern about provoking recurrence of disease. Single dose depomedroxyprogesterone acetate (MPA) 400 mg IM has previously been shown (Loprinzi CL, et al. J Clin Oncol 2006;24:1409) to be the most effective non-estrogen therapy available for menopausal hot flashes (HF) but long-term evidence of safety in survivors of ER+BC is lacking. Methods Consecutive patients previously diagnosed with ER+BC who received MPA for HF between January 2007 and December 2012 were retrospectively identified in the breast cancer patient database at Mayo Clinic Arizona. Medical records were audited for breast cancer outcomes in these cases and in contemporaneous control patients with ER+BC who did not receive MPA, matched for age, stage of disease, and year of diagnosis. Statistical comparisons of local-regional recurrence and event-free survival were performed. Results 92 patients who received MPA were identified and matched 1:1 with contemporaneous controls. Median follow-up duration was 5.7 years in cases and 4.5 years in controls. Estimated local-regional recurrence free survival at 10 years was 85% (95% CI, 72-100%) in cases and 95% (95% CI, 86-100%) in controls. Matched pairs hazard ratio was 1.0 (95% CI, 0.06-16.0) for local-regional recurrence free survival. Estimated event-free survival at 10 years was 81% (95% CI, 69-97%) in cases and 76% (95% CI, 64-92%) in controls. Matched pairs hazard ratio was 0.38 (95% CI, 0.10-1.41) for event-free survival. The majority (77%) of case patients experienced satisfactory relief of hot flashes from MPA injection. Conclusion In this retrospective case-control study we were unable to identify a detrimental effect of MPA therapy for HF in survivors of ER+BC. MPA may be acceptable for management of HF in this population. Citation Format: Ertz-Archambault N, Rogoff L, Kosiorek H, Ernst B, Anderson K, Pockaj B, Gray R, Northfelt D. Depomedroxyprogesterone therapy for hot flashes in survivors of ER-expressing breast cancer: Impact on recurrence and survival [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 P1-11-13.

Abstract Introduction: Breast metastases from non-mammary carcinomas are very rare and the molecular characteristics of these metastases have not been studied. Due to the uniqueness of this type of metastases, we evaluated whether there was any molecular or immunologic similarities among the patients with metastases to the breast. Patients and Methods: Fifty-one patients with metastatic carcinomas to the breast (49 female and 2 male) were identified in the files of Caris Life Sciences (Phoenix, AZ). Average age at presentation was 57 years (range, 20-90 years). Immunohistochemistry for PD-L1 was performed using SP142 (Ventana) or 22c3 (DAKO) antibodies; 592 genes sequencing (n=47), DNA microsatellite instability (MSI) (n=6) and total mutational burden (TMB) (n=14) were obtained from a next generation sequencing platform (Illumina). Results: Three most common primary sites were lung (19, 37%), ovary (15, 29%) and fallopian tubes/peritoneum (7, 14%). Ten metastases (20%) demonstrated neuroendocrine differentiation. When 1% cutoff was applied, tumor cells PD-L1 expression was detected in 10 cases (20%), and immune cells expression of PD-L1 in 19 patients (37%). Three cases (6%) had PD-L1 staining in both cancer and infiltrating immune cells. 50% of the cases exhibited mutations in TP53 gene, while other mutated genes followed the pathways typically seen in their primary sites (e.g. VHL in renal carcinomas, BRCA1 in fallopian tube carcinoma). Total mutational burden was high (≥10 mutations/Mb) in five out of 14 successfully analyzed cases (36%) and no case (0%) exhibited high microsatellite instability. Interestingly, estrogen receptor status was positive in 13/49 patients (26.5%) including 12 adenocarcinomas originating from ovary, Fallopian tube or peritoneum and one duodenal neuroendocrine. No tumor was HER2 positive. Conclusions: Metastases to the breast proved to be heterogeneous but there were molecular, endocrine and immunologic findings that yielded information that could be used to make therapeutic decisions which including the use of new immunotherapeutic strategies. Citation Format: Gatalica Z, Senarathne W, Vranic S, Pockaj BA. Profiling of metastatic carcinomas to the breast for the biomarkers of immuno-oncology therapy [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 P3-10-27.

Abstract OBJECTIVE: Neoadjuvant endocrine therapy (NET) is increasingly used for the treatment of low and intermediate grade, hormone receptor positive, HER2 negative breast cancer. Several MRI phenotypes that may predict response to neoadjuvant chemotherapy (NAC) have been identified, but little data exists for phenotypes associated with response to NET. This study analyzed imaging phenotypes for all patients treated with NET with the aim to identify specific features that can be predictive of response to therapy. MATERIALS AND METHODS: The study was retrospective and included 21 patients with clinical stage I, II, and III breast cancer. The tumors were grade 1 or 2, estrogen receptor (ER) positive in >20% of cells, and HER2 non-amplified. MRI examinations were performed in all women before NET. MRI interpretation included mass shape, non-mass enhancement (NME) pattern, background parenchymal enhancement, and MRI phenotype (I well-defined unicentric mass; II well defined multilobulated mass; III area enhancement with nodularity; IV area enhancement without nodularity; V septal spreading). Type of neoadjuvant endocrine therapy included: tamoxifien alone, an aromatase inhibitor (AI) alone, AI + ovarian suppression, and AI + a non-chemotherapeutic agent. Patients received NET for a total duration ranging between 3 - 6 months, with one patient receiving therapy for 18 months. Clinically meaningful response was defined as stable or decreased tumor size by clinical exam and confirmed at resection by comparing final pathologic T stage with clinical T stage. RESULTS: Twenty-one patients were identified. Median age was 62 (range 36-84) years. Most were post-menopausal 17 (81%). Pre-neoadjuvant meadian tumor size on MRI was 3.9 (range 1.0-7.5) cm and comprised T1 3 (14.3%), T2 8 (38.1%), T3/4 10 (47.6%). Pre-treatment N stage was N0 14 (66.7%), N1 7 (33.3%) and pre-NET stage was I in 3 (14.3%), II in 8 (38.1%), and III in 10 (47.6%) patients. The majority 17 (81%) had some tumor reduction, and 4 (19%) had no response. No one achieved a complete response. Of the 17 responders, 7 (41%) had a good response defined as >25% decrease in tumor size. Median tumor size after NET was 3.1 (range 0.6-11) cm and the distribution of T stage was T1 7 (33.3%), T2 9 (42.9%), and T3/4 5 (23.8%). Eleven of 12 (92%) patients with well-defined phenotypes had a response as compared to 6 of 9 (67%) patients with non-well defined phenotypes. Phenotype was not predictive of a good response to therapy, 4 were in the well-defined phenotype and 3 were in the non-well defined phenotype groups. All 4 non-responders had moderate or marked background enhancement as compared to 5 of 17 responders (p = 0.02). CONCLUSION: A well-defined pre treatment MRI phenotype was significantly predictive of a positive response to NET, while a non well-defined MRI phenotype and higher degree of background enhancement was significantly predictive of negative response to NET. This warrants further prospective evaluation, especially in association with Ki-67 levels. If validated, pre treatment MRI phenotype can be applied in the clinical decision to either initiate NET or referral for upfront surgical resection. Citation Format: Hilal T, Covington M, Pockaj B, Northfelt D, Wu T, Zwart C, Li J, Patel BK. Pre-neoadjuavnt therapy MRI phenotype can predict response to neoadjuvant endocrine therapy [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 P4-02-03.

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.