Citations

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot.Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity.Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. With currently no targeted therapies for TNBC, identification of novel biomarkers to improve chemo-response is vital. Recently, copy number amplifications (CNAs) have been implicated in treatment response in several diseases including breast cancer. Our clinical studies have associated a 5 Mb CNA in TNBC patients with chemo-response, which may elucidate novel biomarkers and treatment strategies for TNBC. Analysis of RNAseq and Whole Exome Sequencing data from 42 TNBC patient tumors enrolled in the BEAUTY Study, identified a 5 Mb CNA on chromosome 8q which associated with pathological complete response following taxane and anthracycline chemotherapy. By performing siRNA knockdown cytotoxicity studies in TNBC cell lines for genes within this amplified region, we identified RDH10 as a potential novel biomarker for increasing chemo-sensitivity. RDH10 is the rate limiting step in the synthesis of all-trans retinoic acid (ATRA). Previous studies have demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways, amplified in TNBC. We discovered overexpression of RDH10 to increase endogenous levels of ATRA, contributing to the degradation of PIN1 and taxane sensitivity. A rescue experiment with PIN1 was able to restore the chemo-resistance phenotype. Similarly, we found treating TNBC cells with ATRA phenocopied RDH10's effect on PIN1 and improved chemo-sensitivity. Furthermore, analyses of chemo-responsive PDX tumors containing the 5 Mb CNA, overexpressed RDH10 and exhibited lower levels of PIN1 when compared to non-responders. In conclusion, RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting RDH10 as a novel biomarker for chemo-response and ATRA as a novel treatment to improve chemo-sensitivity. Citation Format: Jordan Mark Grainger, Jia Yu, Krishna R. Kalari, Peter Vedell, Kevin Thompson, Matthew P. Goetz, Vera J. Suman, Judy C. Boughey, Liewei Wang. Study of copy number amplification and chemo-response in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1928.

9078 Background: A frequently discussed topic at meetings of oncologists is the question of expected clinical outcomes for patients with metastatic melanoma undergoing 1st vs 2nd line systemic therapy. Differing outcomes in these two patient populations could affect interpretation of non-randomized clinical trials involving both patient populations. Some have suggested superior clinical outcome in patients undergoing 2nd line therapy. As there is little data addressing this issue, we sought to answer the question by comparing the clinical outcomes of patients with metastatic melanoma treated on 1st vs 2nd line therapy across clinical trials conducted at our institution. Methods: Data were collected from 10 phase II clinical trials for patients with stage IV melanoma for which Mayo Clinic was the data center. The 10 trials included three categories of treatments: cytotoxic chemotherapy (4), cancer vaccines (4), and biologic agents (2). In all studies, eligibility criteria required: stage IV melanoma, life expectancy >3 months, reasonable hematology and serum chemistry laboratory results, and an ECOG performance status of ≤2. Cox proportional hazards models were fit to assess the relationship between patients' “therapy” status (1st vs 2nd line) and time to events, both overall survival (OS) and progression free survival (PFS), for each treatment category. Results: We identified 318 unique eligible patients across 10 trials. Removed from the analysis were 55 patients (ocular melanoma and/or metastases involving the central nervous system) leaving 263. Cox proportional hazards results demonstrated no differences in PFS or OS for 1st vs 2nd line patients for either “chemotherapy” or “vaccine” treatment regimens. However, patient treated on “biologic” trials as 1st line therapy appeared to demonstrate a PFS advantage over 2nd line treatments (HR=1.98, p-value=0.02). There was a suggestion of an OS benefit for 1st line patients in this category, however, the relationship was not significant (HR=1.77, p=0.07). Conclusions: The presented data suggest that there is no PFS/OS difference in stage IV melanoma patients receiving 1st vs 2nd line therapy (no PFS/OS advantage to patients treated in 2nd line vs. 1st line). No significant financial relationships to disclose.

9055 Background: There is increasing evidence that paclitaxel and carboplatin are clinically active in the treatment of metastatic melanoma (MM). Nab-P is an albumin-bound paclitaxel with ability to bind SPARC (secreted protein acid rich in cysteine), that is overexpressed in MM and associated with poor prognosis. This study explores the clinical activity of the combination of nab-P and C in patients (pts) with stage IV melanoma and SPARC correlatives. Methods: A parallel phase II trial was conducted in pts with unresectable stage IV melanoma, who were either chemotherapy naïve (CN) or were previously treated (PT). A treatment regimen consisting of nab-P (100 mg/m2) and C (AUC 2) was administered on days 1, 8, and 15 of a 28 day cycle. The primary aim of this study was to assess whether tumor response rate (CR + PR by RECIST) was ≤15% vs ≥35% in the CN group and ≤5% vs ≥ 20% in the PT cohort. Major eligibility criteria: ≥18 years of age, ECOG PS ≤2, adequate organ function, platinum or taxane naive, peripheral neuropathy < grade 2, and no untreated brain metastasis; no pregnant and/or nursing women. Tumor tissue was tested for SPARC and level 3 immunohistochemical staining was considered positive. Results: 76 pts (41-CN and 35 PT) enrolled from 11/2006 - 7/2007, 3 pts (2-CN, 1-PT) cancelled prior to starting treatment. The median number of cycles administered was 4 (range 1–18-CN and 1–10-PT). There were 11 (28.2%) confirmed responses (1 CR and 10 PRs) in the CN cohort (90% CI: 16.7–42.3%) and 3 (8.8%) confirmed responses (3 PRs) in the PT cohort (90% CI: 2.5–21.3%). Median PFS was 4.5 months (CN) and 4.1 months (PT). Median OS was 11.1 months (CN) and 10.9 months (PT).The most common severe toxicities in both groups (CTCAE ≥ grade 3) included neutropenia, thrombocytopenia, neuro-sensory, fatigue, nausea, and vomiting. PFS was not affected by SPARC positivity; however, based on limited data there is some evidence that OS may be longer with tumoral SPARC positivity (10.0 vs 12.8 mo; SPARC negative vs SPARC positive). Conclusions: The weekly combination of nab-P and C appears to be well tolerated with promising clinical activity as front line or salvage therapy in pts with MM. SPARC positivity may be associated with improved OS. No significant financial relationships to disclose.

e20013 Background: Recent evidence suggests that patients with advanced cancer exhibit an underlying anti-tumor immune response that is continuously attenuated by regulatory T cells (Treg). Depletion of Treg using conventional chemotherapy may be feasible and clinically beneficial. C-reactive protein (CRP) is reported to rise/fall with initiation/termination of the immune response, and may be a surrogate for the periodicity of Treg proliferation that could be utilized for timing of anti-proliferative therapy. Timed delivery of chemotherapy therapy based on CRP cycling may deplete Treg and yield clinical benefit. Methods: We conducted a pilot clinical for 12 patients with metastatic melanoma who underwent serial CRP measurements (every 2–3 days) for 2 weeks. The CRP oscillation cycle was identified and chemotherapy with temozolomide (200mg/m2 for 5 days, every 28 days) was initiated at the estimated peak of plasma CRP. Patients were evaluated for clinical and immune response endpoints every 8 weeks until progression. Results: All 12 patients (median age 61; 4 female; 7 with M1c disease) exhibited oscillating CRP levels with an average periodicity of 7.8 days. Only 11 patients were treated (1 patient had rapid tumor progression). The two patients who remain progression-free for >2 years (1 PR, 1 CR), were treated in the pre-peak section of the CRP cycle, distinctly separate from the other patients treated post CRP-peak (all progressed <5 months). Presented are peripheral blood immunological laboratory correlates (cellular and cytokine) to CRP oscillation. Conclusions: These data suggest that patient clinical outcome may be dependent on the timing of therapy relative to an individual patient's immune response cycle and outline the dynamic equilibrium of systemic immune homeostasis in patients with advanced melanoma. Further investigations of these observations are under way. [Table: see text]

3521 Background: Systemic therapies have had little impact on the outcomes of patients with advanced differentiated thyroid cancers. Methods: A three-outcome one-stage Phase II trial was conducted to assess the anti-tumor activity and toxicities of the orally bioavailable VEGF/tyrosine kinase inhibitor pazopanib (800 mg daily) in patients with advanced and progressive radioiodine-insensitive differentiated thyroid cancers. Up to 2 prior therapies were allowed, with measurable disease required. Design: at the 0.10 significance level, there would be a 90% chance of detecting a RECIST response rate of >20% given a true response rate of >5%; with the regimen considered promising if >4 confirmed responses observed. Results: From February to November 2008, 32 patients (53% male) aged 23–79 years (median: 63 years) were enrolled. Common sites of metastases were: lung (100%), nodes (52%), and bone (39%). Measurement data are available for 26 patients, with the median number of cycles administered thus far 4 (range: 1–8, total: 101). Overall, therapy has been well tolerated. Six patients (23%) required dose reduction due to: grade 2+ ALT (3 pts), grade 3 mucositis (1 pt); grade 3 diarrhea and dehydration (1 pt), and grade 3 abdominal pain (1 pt.). Other serious toxicities included grade 3 colon perforation and grade 3 chest pain (1 pt). No thyroglobulin antibody (TGA) negative patient has become TGA positive, and 11 of 16 patients (69%) with initially elevated thyroglobulin levels experienced a decline in thyroglobulin of >50%. Five RECIST partial responses have been confirmed to date (19%). Two patients are alive with disease progression and another has died from disease progression. Conclusions: Pazopanib appears to have both a favorable toxicity profile and promising clinical activity in patients with advanced and progressive differentiated thyroid cancers. Supported in part by NCI CA15083 and CM62205. No significant financial relationships to disclose.

550 Background: Ductal carcinoma in situ (DCIS) is a heterogeneous group of non-invasive cancers with varying propensity for recurrence. There are no validated markers that identify patients at risk for invasive recurrence following lumpectomy. HOXB13/IL17BR (H/I) is expressed in both DCIS and invasive cancer (Ma Cancer Cell. 2004) and associated with recurrence in stage I cancer. A 5-gene molecular grade index (MGI) distinguishes outcome in grade II disease (Ma Clin Cancer Res. 2008). We sought to determine whether H/I or MGI was associated with invasive recurrence in resected DCIS. Methods: We identified patients at Mayo Rochester who underwent lumpectomy ± radiation for DCIS between 1988 and 2001 and performed a nested case/control study. Cases were patients with ipsilateral invasive recurrence, matched to controls based on age, surgery date, length of follow-up, and adjuvant radiation. DCIS was macrodissected from paraffin sections and RNA extracted to obtain H/I and MGI RT-PCR profiles. The association of H/I and MGI with case-control status was assessed with conditional logistic regression; results reported are odds ratios (OR) with 95% confidence intervals (CI). The association of H/I and MGI as continuous variables with nuclear grade (low, intermediate, high) was estimated with Spearman's rank correlation. Results: 427 patients underwent lumpectomy for DCIS, and 33 were identified as possible cases. Histologic review excluded 8 (non-sufficient tissue or DCIS recurrence without invasion). 25 cases were matched to 48 controls (2 controls for 23 cases, 1 control for 2 cases). Both H/I (r = 0.43, p = 0.0001) and MGI (r = 0.41, p = 0.0004) were significantly correlated with nuclear grade. Positive H/I was associated with trend towards higher recurrence (OR 1.9; p = 0.22) that was most pronounced in cases/controls (15/28) treated with lumpectomy alone (OR 3.3; CI: 0.8–13.2, p = 0.09). Similarly, positive MGI was associated with a trend towards higher recurrence risk in patients treated with surgery alone (OR 2.0; CI: 0.6–6.4, p = 0.27). Conclusions: These data suggest that both HOXB13/IL17BR and MGI can identify a subset of patients with surgically resected DCIS who are at risk for invasive recurrence. Further studies in larger cohorts are needed to confirm these findings. [Table: see text]

Abstract Asparagine‐linked glycosylation 13 homolog ( ALG13 ) encodes a nonredundant, highly conserved, X‐linked uridine diphosphate (UDP)‐ N ‐acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N‐linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13‐CDG. Twenty‐four previously reported ALG13‐CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13‐CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13‐deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13 . This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.

Introduction: Multikinase inhibitors have clinical activity in radioactive iodine refractory (RAIR) differentiated thyroid cancers (DTCs) but are not curative; optimal management and salvage therapies remain unclear. This study assessed clinical effects of pazopanib therapy in RAIR-DTC patients with progressive disease, examining in parallel biomarker that might forecast/precede therapeutic response. Methods: Assessment of responses and toxicities and of any association between thyroglobulin (Tg) changes cycle 1 and RECIST (response evaluation criteria in solid tumors) response to pazopanib therapy were prospectively undertaken in Tg antibody negative RAIR-DTC patients. RECIST progressive metastatic disease <6 months preceding enrollment was required. With a sample size of 68 (assuming 23 attaining partial response [PR]), there would be 90% chance of detecting a difference of >30% when the proportion of patients attaining PR whose Tg values decrease by >50% is >50% cycle 1 (one-sided α = 0.10, two sample test of proportions). Mean corpuscular volume (MCV) change or mutational status or pretreatment were also explored as early correlates of eventual RECIST response. Results: From 2009 to 2011, 60 individuals were treated and evaluated; (one additional patient withdrew; another was found ineligible before therapy initiation); 91.7% had previous systemic therapy beyond RAI. Adverse events included one death (thromboembolic) deemed possibly pazopanib associated. Twenty-two confirmed RECIST PRs resulted (36.7%, confidence interval; CI [24.6–50.1]); mean administered 4-week cycles was 10. Among 44 fully accessible patients, the Tg nadir was greater among the 20 attaining PR (median: −86.8%; interquartile range [IQR]: −90.7% to −70.9%) compared with the 28 who did not (median: −69.0%; IQR: −78.1% to −27.7%, Wilcoxon rank-sum test: p = 0.002). However, the difference in the proportion of PRs among those whose Tg fell ≥50% after cycle 1 versus those that did not were not significantly correlated (−23.5% [CI: −55.3 to 8.3]; Fisher's exact test p -value = 0.27). RECIST response was also not correlated with/predicted by early MCV change, receipt of prior therapy, or tumor mutational status. Conclusions: This trial prospectively confirmed pazopanib to have clinical activity and manageable toxicities in patients with progressive RAIR-DTC. Response to pazopanib, however, was not robustly forecast by early associated changes in Tg or MCV, by prior therapy, or by tumor mutational status. ClinicalTrials.gov NCT00625846.

e22020 Background: The combination of nab-paclitaxel (NP) and bevacizumab (BEV) in patients with MM has shown promising clinical activity. AB160 is a 160 nm nano-immunoconjugate of NP nanoparticles non-covalently coated with BEV for targeted delivery into high VEGF expressing tissues. Preclinical data showed that AB160 improved tumor targeting/ tumor inhibition more than NP followed by BEV. Methods: A 3+3 phase I trial was conducted in patients (pts) with MM who had prior systemic treatment for metastatic disease to determine the maximum tolerated dose of AB160 administered intravenously on days 1, 8 and 15 of a 28-day cycle. Dose level 1 (DL1) was 125 mg/m2 NP /50 mg/m2 BEV. Dose limiting toxicities (DLT) included grade (G) 4 neutropenia or anemia, PLT < 25,000, serum creatinine ≥ 2 times baseline, G2-4 neurologic toxicity or G3-4 non-hematologic toxicities. Tumor evaluations (RECIST) were conducted every 8 weeks. Treatment continued until progression or intolerability. Results: 21 pts (11 ♀) aged 36-78 years old were enrolled. One of the first 3 pts on DL1 developed a G2 colonic perforation; this was considered a DLT. One of the next 3 pts on DL1 had a DLT: G4 neutropenia. Of the 3 pts on DL-1 (100 mg/m2 NP/40 mg/m2 BEV), 2 had no DLTs and 1 died of sepsis after C1D1 dose. Enrollment was suspended until an amendment modifying the eligibility criteria was approved by the IRB. The trial reopened. One of the 4 pts on DL-1 and 1 of the 5 pts on DL1 had a DLT: G3 pain and G3 fatigue, respectively. Enrollment ended after 2 of the 3 pts on DL2 (150 mg/m2 NP/ 60 mg/m2 BEV) developed G4 neutropenia. Thus, MTD is DL1. A median of 3 cycles was administered. Treatment ended due to progression (9), intolerability (9), refusal (2) and death (1). There were no objective tumor responses. Common G3-4 toxicities were: neutropenia (33%) and thromboembolic events (19%). Conclusions: AB160 was found to have insufficient clinical benefit in patients with previously treated MM to justify further development. However, parallel phase I testing in gynecologic cancers suggests clinical benefit (abstract #300225). Clinical trial information: NCT02020707.