Citations

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.

463 Background: The genomic assessment of cancer has been revolutionized by next-generation sequencing and is increasingly being applied in the clinic to guide therapeutic decision-making. Time to reporting of results, specimen quantity, and analyte quality have constrained initial clinical applications to gene panels and whole exome based strategies. Methods: Patients underwent surgical resection, excisional or core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing and RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients with advanced, treatment-refractory cancers of whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in the CLIA enabled phase. Upon availability of the initial report, identified targets of putative therapeutic relevance were then prioritized by the CGB in 22/27 patients (81%) for subsequent CLIA validation. Eleven patients have been treated with genomically selected therapy with partial response in 3/10 assessed patients. A testicular cancer patient had aberrations in two testes specific genes, a copy number gain in TSSK6 and a novel gene fusion between thyroid hormone receptor associated protein 3 (THRAP3) and Tektin 2 (TEKT2). Additionally, a case of papillary renal cell carcinoma had an amplification of YAP1 and a previously unreported P287T mutation in CCND1, suggesting potential benefit with a CDK4/6 inhibitor. Treatment is ongoing and results will be reported. Conclusions: Integrated whole genome analysis in a CLIA setting is feasible. Integration of SNV, copy number and transcriptional data may allow for selection of putative driver genes to enhance targeted therapy decisions. Barriers for future broader implementation include the need for reduced time from biopsy to report and availability of therapies.

572 Background: There is limited data regarding chemotherapy dosing in obese mCRC patients. Methods: A retrospective, single institution, cohort study of mCRC patients receiving mFOLFOX6 (fluorouracil (5-FU) bolus omitted) was conducted between January 2002 and January 2011. Patients were stratified into: obese (BMI > 30) full dose, obese dose reduced (oxaliplatin (OX) and/or 5-FU), non-obese (BMI 18-29.9) full dose, and non-obese dose reduced. The primary endpoint was incidence of grade 3/4 myelosuppression within the first 12 wks of therapy. Secondary endpoints included incidence of any grade diarrhea and neuropathy, progression free (PFS) and overall survival (OS). Results: Of the 236 patients (72 obese, 164 non-obese), 15 obese and 37 non-obese received a dose reduction. Grade 3/4 myelosuppression occurred in 0 obese OX dose reduced, 3 (7.5%) obese full dose, 1 (8.3%) non-obese dose reduced, and 7 (7.8%) non-obese full dose patients (p=0.93). Grade 3/4 myelosuppression occurred in 0 obese 5-FU dose reduced, 3 (7.5%) obese full dose, 2 (12.5%) non-obese dose reduced, and 6 (7%) non-obese full dose patients (p=0.8). There were no statistically significant differences in the incidence of neuropathy or diarrhea between groups. PFS was longer in OX full dose patients (obese dose reduced, 8.8 mos; obese full dose, 10 mos; non-obese dose reduced, 3.7 mos; non-obese full dose, 10.4 mos; p=0.0094). No difference in PFS was found with 5-FU dosing (p=0.0615). OS favored OX full dose patients (obese dose reduced, 1.6 yrs; obese full dose, 2.4 yrs; non-obese dose reduced, 0.7 yrs; non-obese full dose, 2 yrs; p=0.0006) and 5-FU full dose patients (obese dose reduced, 1.6 yrs; obese full dose, 2.2 yrs; non-obese dose reduced, 1.6 yrs; non-obese full dose, 1.9 yrs; p=0.0287). Conclusions: There is no difference in the incidence of grade 3/4 myelosuppression, diarrhea or neuropathy in obese versus non-obese patients, dose reduced or not. OX dose reductions were associated with reduced PFS and OS. 5-FU dose reductions were associated with reduced OS. Our study supports ASCO recommendations against dose reductions in obese patients receiving chemotherapy.

238 Background: Dual inhibition of the EGFR pathway using erlotinib and the monoclonal antibody panitumumab results in greater antitumor activity in preclinical pancreatic cancer models. A trial evaluating this approach was conducted. Methods: A safety analysis was initially performed with 6 patients at P 4 mg/kg every two weeks, E 100 mg daily, G 1000 mg/m2 weekly of a 28-day cycle. PGE was well tolerated with a grade 3 fatigue as only dose-limiting toxicity. Patients (pts) were then randomized to either PGE or GE alone. Eligibility included: no prior chemo, ECOG PS 0-1, bilirubin <2x UL, AST <2.5x UL. CT scans every 2 cycles. Results: 93 pts (46-GE; 47-PGE) were randomized from January to August, 2010. Median age 61 years and 65% male. ECOG PS 0 (51%) and PS 1 (49%). No significant differences in baseline characteristics observed between treatment arms. 81 pts evaluable for AEs; 37 (46%) had a maximum grade 3 (at least possibly related to treatment), and 12 (15%) maximum grade 4 or 5. Treatment arms similar with respect to the overall AEs with exception grade 1-3 acneiform rash, GE (65%) vs. PGE (85%). Commonly occurring grade 4 AEs (at least possibly related) consisted of thrombosis (3 pts) and thrombocytopenia (2 pts). Two grade 5 events occurred in PGE arm—duodenal hemorrhage (possibly related) and pancolitis with multisystem failure (unlikely). An additional PGE pt died 30 days off study (grade 5 CHF, possibly). 54 patients (67%; 31-GE, 23-PGE) ended treatment; 38 (70%) for disease progression (24-GE; 14-PGE). Other reasons for treatment discontinuation: AEs (9; 17%), pt refusal (4; 7%), death on study (2; 4%), other (1). In 81 pts with at least 1 cycle of chemotherapy, the median PFS for GE 2.0 months and 3.3 months for PGE. Accrual for the study was recently completed and overall and 6-month survival data will be presented. Conclusions: This study involved an initial run-in design to define tolerable doses of dual EGFR inhibitors, PGE, in pancreatic cancer patients. Although preliminary, the randomized phase II portion has been completed and differences between GE and PGE in terms of relevant clinical outcomes, PFS, are observed. No significant financial relationships to disclose.

A common complication associated with aging is the stiffening of skeletal muscles. The purpose of this study was to determine the ability of magnetic resonance elastography (MRE) to study this phenomenon in vivo. Twenty female subjects were included in the study with an age range of 50 to 70 years. Shear modulus was calculated for the tibialis anterior of each subject. There was not a significant relationship between age and shear modulus. However, three subjects had abnormally high values and were among the oldest subjects tested. There was a significant relationship between age and tissue stiffness homogeneity. More research is needed to determine whether the changes seen here are reflective of increased tissue cross-linking or related to reduced muscle quality. However, MRE shows promise as a tool to study aging-related muscle stiffness changes or to evaluate treatments to counteract these changes.