Citations

Abstract Background: Tumorigenesis involves both genetic and epigenetic changes. Epigenetic alterations are reversible and are promising cancer therapeutic targets. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferase inhibitor, is FDA approved for hematological malignancies. However, the effect of decitabine in breast cancer is not completely understood. Previous reports indicated that one decitabine mechanism involves regulation of protein levels for DNMT1, the major DNA methyltransferase that methylates hemimethylated CpG di-nucleotides in DNA. However, the E3 ligase involved in this process has not been identified. Whether decitabine also regulates DNMT3A and 3B in a similar fashion remains unclear. Therefore, our goals were to 1) understand mechanisms underlying decitabine action, 2) test the antitumor activity of decitabine in breast cancer models and 3) identify biomarkers associated with response to decitabine. Methods and Results: Western blots of breast cancer cell lines showed that DNMT1, DNMT3A, and DNMT3B protein levels decreased following decitabine treatment without a reduction in mRNA levels. Bioinformatic analysis of DNA methyltransferase sequences revealed a potential TRAF6 binding motif, and the interaction with TRAF6 (TNF receptor-associated factor 6) was confirmed by IP. TRAF6 functions as an E3 ligase. To determine whether TRAF6 might be the E3 ligase responsible for the degradation of DNMTs after decitabine treatment, we knocked down TRAF6 by RNA interference or knocked out the TRAF6 gene by CRISPR/Cas9. Down regulation of TRAF6 attenuated DNMT ubiquitination and increased DNMT protein levels, suggesting that TRAF6 might mediate proteasome-dependent degradation of all three DNMTs. This was further confirmed by reconstituting the knockout cells with WT and a TRAF6-C70A mutant, followed by assessing DNMT protein levels. Global DNA methylation was also increased after TRAF6 depletion and was confirmed in TRAF6 knock out cells in which DNMT levels were unaffected by decitabine. Cell cytotoxicity and colony forming assays showed that TRAF6 knockout cells were resistant to decitabine, suggesting that a major decitabine mechanism of action is through the regulation of TRAF6 which, in turn, degrades DNMTs, leading to decreased global methylation. Finally, decitabine significantly induced TRAF6 at both mRNA and protein levels, a process that might create positive feedback leading to increased degradation of DNMT proteins upon decitabine treatment. Based on these results, we further hypothesized that levels of the three DNMTs might influence decitabine response. Using 18 breast cancer patient derived xenograft (PDX) models, we found a wide range of DNMT protein levels regardless of ER/HER2 status. DNMT levels in the PDX models were directly associated with sensitivity to decitabine treatment, confirming our hypothesis. Conclusion: Our data showed that decitabine might be an effective agent for treating breast cancer and revealed a novel mechanism underlying decitabine treatment. Baseline DNMT protein levels may serve as a biomarker for predicting decitabine drug response. Citation Format: Yu J, Qin B, Boughey JC, Moyer AM, Visscher DW, Sinnwell JP, Yin P, Thompson KJ, Docter TJ, Kalari KR, Suman VJ, Wieben ED, Felten SJ, Conners AL, Jones KN, McLaughlin SA, Copland JA III, Moreno Aspitia A, Northfelt DW, Gray RJ, Ingle JN, Lou Z, Weinshilboum R, Goetz MP, Wang L. Regulation of DNA methyltransferases via TRAF6 determines breast cancer response to decitabine. [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 P3-07-51.

Abstract Background: Both HR deficiency and BRCA mutation status predict response to platinum-based therapy and BRCA mutation status predicts docetaxel resistance. However, the association of either biomarker with response to the individual elements of either AC or taxanes (T) is unknown since T is commonly given concomitantly with or after anthracyclines (A). We evaluated the association of HRD and BRCA mutation status with response to neoadjuvant weekly T followed by AC or (F)EC in high-risk breast cancer. Methods: We studied 140 high risk Stage I-III breast cancer patients (pts), enrolled in the breast cancer genome guided therapy study (BEAUTY), obtaining biopsies for DNA/RNA sequencing and MRI imaging to assess response to neoadjuvant weekly T (+trastuzumab+/-pertuzumab for HER2+ disease) followed by AC or (F)EC. Germline BRCA status and HR status of tumor samples (Myriad laboratories) were obtained. HR deficient tumor was defined as HRD score ≥42 or BRCA mutation. MRI response by changes in tumor size after 12 weeks of T was classified by WHO criteria. pCR was defined as ypT0/Tis ypN0. Both MRI response after T and pCR (after T and AC) were examined in terms of germline BRCA mutation (gBRCAmut vs. gBRCAwt) and tumor HR deficiency. Results: Of 140 pts enrolled, 8 withdrew consent and 2 carboplatin treated pts were excluded. Germline data were available for 124/130 pts. 12 patients had BRCA deleterious germline mutations (4 BRCA1, 8 BRCA2). MRI partial (PR)/complete response (CR) rate to T was 47.3% (95% CI: 37.8-57.0%) in the BRCAwt group and 66.7% (95% CI: 34.9-90.1%) in the BRCAmut group. No MRI CR's were observed in BRCA1 mut pts. In contrast, pCR rate was 50% in the 12 gBRCAmut pts (95% CI: 21.1-78.9%) and 31.3% in the 112 gBRCAwt pts (95% CI: 22.8-40.7%). HR deficiency status has thus far been determined for 74 pts: 26 pts have HD deficient tumors: 18 TNBC, 5 Luminal B, 2 ER-/HER2+; and 1 ER+/HER2+. Determination of HR deficiency is ongoing and will be reported for the full cohort in terms of 12 week MRI response to T and pCR to T+AC. HR deficientMolecular Subtypeyes (%)no (%)TBD (%)Luminal A0/112/11 (18.2)9/11 (81.8)Luminal B5/37 (13.5)13/37 (35.1)19/37 (51.3)Luminal NOS0/21/2 (50)1/2 (50)ER+/Her2+1/17 (5.8)14/17 (82.4)2/17 (11.8)ER-/Her2+2/20 (10)11/20 (55)7/20 (35)Triple Negative18/43 (41.9)6/43 (18.6)17/43 (39.5)germline BRCA statusMRI partial response after T (%)MRI complete response after T (%)pCR after T&AC (%)BRCA11/4 (25)0/42/4 (50)BRCA25/8 (62.5)2/8 (25)4/8 (50)BRCAwt35/112 (31.3)18/112 (16.1)35/112 (31.3) Conclusion: In the setting of neoadjuvant weekly T followed by AC, pCR rates were non-significantly higher in pts with BRCA1 mutations. While we observed no overall association between BRCA mutation status and response rates to taxanes; nearly all MRI responses to taxanes (partial and complete) were observed in the BRCA2 group. Prospective studies are needed to validate these findings and to determine whether BRCA status can be used to select therapy. HR deficiency is uncommon in luminal A and HER2+, frequent in TNBC, and the association of HRD with both MRI response to taxanes and pCR will be reported at the meeting. Citation Format: Boughey JC, Kalari KR, Suman VJ, McLaughlin SA, Moreno Aspitia A, Moyer AM, Northfelt DW, Gray RJ, Vedell PT, Tang X, Dockter TJ, Jones KN, Felten SJ, Conners AL, Hart SN, Visscher DW, Wieben ED, Ingle JN, Hartman A-R, Timms K, Elkin E, Jones J, Wang L, Weinshilboum RW, Goetz MP. Role of germline BRCA status and tumor homologous recombination (HR) deficiency in response to neoadjuvant weekly paclitaxel followed by anthracycline-based chemotherapy. [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 P3-07-29.

Abstract Background: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer. [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 P2-05-21.

Abstract Background: Nal-IRI (MM-398, nanoliposomal irinotecan) is designed for extended circulation relative to free irinotecan and to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with response to therapy. In phase I studies of nal-IRI, activity has been shown in metastatic breast cancer (MBC), pancreatic and colorectal cancer. Ferumoxytol (FMX) is an iron-oxide superparamagnetic nanoparticle that has been used off-label for its MRI contrast properties. FMX has long-circulating pharmacokinetics and is taken up by TAMs with similar distribution patterns to nal-IRI in preclinical models. A single site pilot study established the feasibility of performing quantitative FMX MRI. Thirteen patients with advanced cancer (3 with ER/PR+ MBC) were imaged with FMX MRI and treated with nal-IRI. Median tumor lesion FMX uptake in the pilot study was 32.6 and 34.5 μg/mL at 1 h and 24 h, respectively. Lesions with FMX uptake above the median were associated with greater reductions in tumor size following treatment with nal-IRI as determined by CT lesion measurements. The relationship between FMX levels in tumor lesions and nal-IRI activity may serve as a potential biomarker for nal-IRI deposition and response in solid tumors. This study has been expanded to include additional MBC patients to further evaluate the technical feasibility of FMX MRI at multiple study sites, and to evaluate activity of nal-IRI in patients with MBC. Trial Design: Three cohorts of 10 patients with MBC in the following categories will be enrolled: ER and/or PR positive/HER2-negative, triple negative (TNBC) and MBC with brain metastases. An imaging phase will be followed by a treatment phase. The imaging phase consists of a baseline MRI scan, FMX infusion, and follow-up MRI scans at 1-4 and 24 h after infusion. The treatment phase begins 1-6 days after imaging and consists of nal-IRI 80 mg/m2 q2w. A pretreatment biopsy is required for correlative studies. Study Objectives: The primary objective of this multisite expansion is to investigate the feasibility of FMX quantitation in tumor lesions at multiple lesion sites in breast cancer. The secondary objective is to characterize the efficacy of nal-IRI in patients with metastatic breast cancer. Eligibility Criteria: The key inclusion criteria include patients with MBC, ECOG 0 or 1 with adequate bone marrow reserve and no prior topoisomerase 1 inhibitor or anti-VEGF treatment. ER and/or PR positive/HER2-negative and TNBC patients must have had 1-3 prior lines of chemotherapy in the metastatic setting and have at least 2 measurable lesions. Patients with brain metastasis must be neurologically stable and have new or progressive brain metastases after prior radiation therapy with at least one lesion measuring ≥ 1 cm in longest diameter on gadolinium-enhanced MRI. Status: This trial is currently recruiting patients. Citation Format: Sachdev JC, Ramanathan RK, Raghunand N, Anders C, Munster P, Minton S, Northfelt D, Blanchette S, Campbell K, Lee H, Klinz SG, Hendriks BS, Moyo V, Fitzgerald JB, Korn RL. A phase 1 study in patients with metastatic breast cancer to evaluate the feasibility of magnetic resonance imaging with ferrumoxytol as a potential biomarker for response to treatment with nanoliposomal irinotecan (nal-IRI, MM-398). [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 OT3-02-14.

Abstract The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

BackgroundTrastuzumab has substantially improved overall survival and reduced the risk of disease recurrence in patients with human epidermal growth factor receptor type II (HER‐II)–positive breast cancer. However, this benefit may be at the increased risk of cardiotoxicity. We aimed to explore the early subclinical left and right ventricular as well as atrial dysfunction, in trastuzumab‐treated patients with HER‐II–positive breast cancer, using velocity vector imaging.MethodsEchocardiography images were acquired in 50 patients with HER‐II–positive breast cancer undergoing trastuzumab therapy. All patients had baseline and 3–6 months and 12–15 months of follow‐up echocardiograms after initiation of trastuzumab therapy. Subendocardial borders of all the cardiac chambers were traced from the apical views to obtain volumetric and deformation indices.ResultsMean age was 60 ± 13 years. Left ventricular (LV) ejection fraction as well as conventional indices of right ventricular (RV) function did not change with trastuzumab. The RV peak systolic global longitudinal strain (GLε) significantly decreased (−24.53 ± 6.03 vs. −21.28 ± 5.11 vs. −21.84 ± 5.15, baseline vs. first and second follow‐ups, P = 0.01). LV peak systolic GLε was reduced by 1.19 at early follow‐up (P < 0.05). Left atrial reservoir and booster pump functions as well as right atrial reservoir function were reduced through follow‐up as well.ConclusionsThe RV exhibited greater change in strain after trastuzumab treatment when compared to the LV. Atria function was reduced by trastuzumab as well. The repercussion of these findings and their potential implication will warrant further study.

Abstract Purpose: This phase II trial evaluated the efficacy and safety of cixutumumab, a human anti–insulin-like growth factor receptor 1 (IGF-1R) monoclonal IgG1 antibody, and explored potential biomarkers in postmenopausal women with hormone receptor–positive breast cancer. Experimental Design: Patients with hormone receptor–positive breast cancer that progressed on antiestrogen therapy received (2:1 randomization) cixutumumab 10 mg/kg and the same antiestrogen (arm A) or cixutumumab alone (arm B) every 2 weeks (q2w). Primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS) and safety. Correlative analyses of IGF-1R, total insulin receptor (IR), and IR isoforms A (IR-A) and B (IR-B) expression in tumor tissue were explored. Results: Ninety-three patients were randomized (arm A, n = 62; arm B, n = 31). Median PFS was 2.0 and 3.1 months for arm A and arm B, respectively. Secondary efficacy measures were similar between the arms. Overall, cixutumumab was well tolerated. IGF-1R expression was not associated with clinical outcomes. Regardless of the treatment, lower IR-A, IR-B, and total IR mRNA expression in tumor tissue was significantly associated with longer PFS [IR-A: HR, 2.62 (P = 0.0062); IR-B: HR, 2.21 (P = 0.0202); and total IR: HR, 2.18 (P = 0.0230)] and OS [IR-A: HR, 2.94 (P = 0.0156); IR-B: HR, 2.69 (P = 0.0245); and total IR: HR, 2.72 (P = 0.0231)]. Conclusions: Cixutumumab (10 mg/kg) with or without antiestrogen q2w had an acceptable safety profile, but no significant clinical efficacy. Patients with low total IR, IR-A, and IR-B mRNA expression levels had significantly longer PFS and OS, independent of the treatment. The prognostic or predictive value of IR as a biomarker for IGF-1R–targeted therapies requires further validation. Clin Cancer Res; 22(2); 301–9. ©2015 AACR.

Abstract Introduction: Overexpression and amplification of PD-L1 has been reported in a subset of cancers, including lymphoma, and gastric cancer. We have recently described amplification of chromosome 9p24 targeting PD-L1 in a subset of triple negative breast cancers (TNBC), as well as 1-3% of colorectal carcinomas and glioblastomas. The clinical impact of these amplifications is not known. Methods: Tumor samples from 41 subjects with TNBC were evaluated for copy number aberrations. We performed DNA-based flow cytometry to sort nuclei from diploid, tetraploid, and aneuploid populations of tumor cells. The genomes of each sorted tumor cell population were then interrogated with oligonucleotide CGH arrays. Copy number intervals were identified and their genomic boundaries mapped using a step gram algorithm. Clinical data was available on 36 of 45 (80%) patients. Results: A recurring top ranked high level (log2ratio &gt;1) amplicon that spanned 9p24.1 was detected in 12/41 (29.3%) of TNBCs. In contrast this amplicon was absent in ER+ (n = 8) and Her2+ (N = 15) breast tumors. The shortest region of overlap (SRO) spanned 777 kb and always included the PD-1 ligands PD-L1, PD-L2, and Janus kinase 2 (JAK2) loci, termed the “PDJ” amplicon. Additional genes in this amplicon include RCL1, MIR101-2, INSL6, INSL4, RLN2, RLN1, and C9orf46. The height of the PDJ amplicon included mean log2ratios of &gt;4, consistent with genomic drivers such as Her2 and Myc. Patients with PD-L1 amplification (n = 8) were noted to have larger tumors (mean 3.9 vs. 1.9 cm, p = 0.04) and a higher incidence of lymph node metastases (75% vs. 26%, p = 0.01). Lymphocytic infiltration was only noted in 4 patients, all without PDJ amplification. Almost all patients received chemotherapy after definitive surgical therapy. The recurrence rate at 5 years was 62.5% in the PDJ amplified patients, and 25% in the unamplified patients (p = 0.02). Overall survival at 5 years was 37.5% in the PDJ amplified patients, compared with 78.2% in the unamplified patients (p = 0.02). Conclusion: We identified a focal amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 in a significant proportion of TNBCs. The high level of amplification and inverse correlation with survival suggests that the PDJ amplicon is a genetic driver event in these cancers, and warrants confirmation in larger studies. Citation Format: Karen S. Anderson, Christine L. Klassen, Amylou C. Dueck, Ann E. McCullough, Donald W. Northfelt, Mariacarla Andreozzi, Barbara A. Pockaj, Michael T. Barrett. High-level amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with worse DFS and OS in triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2015-2861

AbstractPurpose: Immunotherapy as a treatment for cancer holds the promise of complete and durable tumor remission, yet the immunosuppressive environment created by many tumors, advanced patient age, and previous treatments with cytotoxic agents may limit the approach. The activity of motolimod (VTX-2337), a potent and selective Toll-like receptor 8 (TLR8) agonist, was therefore assessed in the context of advanced, late-stage cancer patients.Experimental Design: The repertoire of mediators induced from human peripheral blood mononuclear cells in response to motolimod was characterized. Translational studies in cynomolgus monkeys elucidated the activity of motolimod on an intact immune system, identified biomarkers of TLR8 activation, and defined the relationship between the pharmacokinetic and pharmacodynamic (PK/PD) response. The PK/PD relationship for motolimod in cancer patients was assessed, compared with preclinical findings, and contrasted with activity in healthy volunteers.Results: In late-stage cancer patients, plasma levels of multiple biomarkers, including IL6, G-CSF, MCP-1, and MIP1-β, increased with increasing motolimod dose. The magnitude and breadth of the biomarker response closely aligned with the response seen in preclinical studies, demonstrating that advanced cancer patients remained responsive to TLR8 activation. In addition, the PK/PD response in cancer patients closely aligned with the activity of motolimod seen in healthy volunteers.Conclusions: Late-stage cancer patients are highly sensitive to TLR8 activation by motolimod. Tumor burden, advanced age, and prior treatment history with cytotoxic agents did not moderate or modify the response predicted by nonclinical studies and confirmed in healthy volunteers. Clin Cancer Res; 21(24); 5445–52. ©2015 AACR.