Abstract
Glycoprotein non-metastatic melanoma B (GPNMB) is a transmembrane protein overexpressed in 30 - 40% of triple negative breast cancer (TNBC) and has shown to be associated with metastasis and ...disease recurrence. An anti-GPNMB antibody drug conjugate, Glembatumumab Vedotin (CDX-011), is currently in Phase II clinical trials for the treatment of metastatic TNBC patients, with promising outcomes. Positron Emission Tomography using radiolabeled antibodies could be advantageous in stratifying patients who may benefit from CDX-011, tracking the biodistribution of CDX-011, and assessing GPNMB expression in vivo. To this end, we radiolabeled the naked antibody, Glembatumumab (CR011), with the positron-emitting 89Zr (half life = 3.3 days). We characterized the stability, affinity, rate of cellular internalization, and specificity of 89Zr-CR011 using various cell-binding assays in human TNBC cell lines. We determined that 89Zr-CR011 is stable in serum solution for up to 5 days, binds specifically to GPNMB+ TNBC cells with high affinity (KD = 16 nM), and internalizes rapidly (50% within 30 - 60 min). We conducted a biodistribution study from 1 - 12 days post administration via tail vein in GPNMB+ MDA-MB-468 xenografts to determine the time point at which we achieve the optimal tumor-to-nontarget ratios. A subset of mice was administered a blocking dose of unlabeled CR011 (100-fold excess, 1 mg/mouse), where we observed a 2.5-fold reduction in 89Zr-CR011 tumor uptake, confirming the specificity of 89Zr-CR011 for GPNMB+ TNBC tumors. PET imaging studies and dosimetry calculations are currently in progress. This preliminary study demonstrates that 89Zr-CR011 may be an excellent companion diagnostic agent for CDX-011 therapy and an essential tool to assess the function of GPNMB in vivo.
Citation Format: Bernadette V. Marquez, Supum Lee, Tibor Keler, Thomas Hawthorne, Jeremy Hoog, Shunqiang Li, Cynthia Ma, Suzanne E. Lapi. Targeting GPNMB with 89Zr-CR011 for PET imaging of triple negative breast cancer. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4209.
Abstract
Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics ...analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (>10,000 proteins) and more than >20,000 phosphosites starting with just 25 micrograms of protein per sample. In order to understand the capabilities and limitations our our approach relative to more conventional deepscale proteomics requiring >10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ locally advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative, microscaled proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a robust proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting.
Citation Format: Shankha Satpathy, Eric Jaehnig, Krug Karsten, Beom-Jun Kim, Alexander Saltzman, Doug Chan, Kimberly Holloway, Meenakshi Anurag, Chen Huang, Purba Singh, Ari Gao, Noel Namai, Yongchao Dou, Bo Wen, Suhas Vasaikar, David Mutch, Mark Watson, Cynthia Ma, Foluso Ademuyiwa, Mothaffar Rimawi, Jeremy Hoog, Samuel Jacobs, Anna Malovannaya, Terry Hyslop, D.R Mani, Charles Perou, George Miles, Bing Zhang, Michael Gillette, Steven Carr, Matthew Ellis. Microscaled proteogenomic methods for precision oncology abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-05.
Next-generation sequencing technologies have provided insights into the biology and mutational landscape of cancer. Here, we evaluate the relevance of cancer neoantigens in human breast cancers. ...Using patient-derived xenografts from three patients with advanced breast cancer (xenografts were designated as WHIM30, WHIM35, and WHIM37), we sequenced exomes of tumor and patient-matched normal cells. We identified 2,091 (WHIM30), 354 (WHIM35), and 235 (WHIM37) nonsynonymous somatic mutations. A computational analysis identified and prioritized HLA class I-restricted candidate neoantigens expressed in the dominant tumor clone. Each candidate neoantigen was evaluated using peptide-binding assays, T-cell cultures that measure the ability of CD8
T cells to recognize candidate neoantigens, and preclinical models in which we measured antitumor immunity. Our results demonstrate that breast cancer neoantigens can be recognized by the immune system, and that human CD8
T cells enriched for prioritized breast cancer neoantigens were able to protect mice from tumor challenge with autologous patient-derived xenografts. We conclude that next-generation sequencing and epitope-prediction strategies can identify and prioritize candidate neoantigens for immune targeting in breast cancer.
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High levels of expression of glycoprotein non-metastatic B (gpNMB) in triple negative breast cancer (TNBC) and its association with metastasis and recurrence make it an attractive target for therapy ...with the antibody drug conjugate, glembatumumab vedotin (CDX-011). This report describes the development of a companion PET-based diagnostic imaging agent using
Zr-labeled glembatumumab (
ZrDFO-CR011) to potentially aid in the selection of patients most likely to respond to targeted treatment with CDX-011.
ZrDFO-CR011 was characterized for its pharmacologic properties in TNBC cell lines. Preclinical studies determined that
ZrDFO-CR011 binds specifically to gpNMB with high affinity (Kd = 25 ± 5 nM), immunoreactivity of 2.2-fold less than the native CR011, and its cellular uptake correlates with gpNMB expression (r = 0.95). In PET studies at the optimal imaging timepoint of 7 days p.i., the
ZrDFO-CR011 tumor uptake in gpNMB-expressing MDA-MB-468 xenografts had a mean SUV of 2.9, while significantly lower in gpNMB-negative MDA-MB-231 tumors with a mean SUV of 1.9.
ZrDFO-CR011 was also evaluated in patient-derived xenograft models of TNBC, where tumor uptake
had a positive correlation with total gpNMB protein expression via ELISA (r = 0.79), despite the heterogeneity of gpNMB expression within the same group of PDX mice. Lastly, the radiation dosimetry calculated from biodistribution studies in MDA-MB-468 xenografts determined the effective dose for human use would be 0.54 mSv/MBq. Overall, these studies demonstrate that
ZrDFO-CR011 is a potential companion diagnostic imaging agent for CDX-011 which targets gpNMB, an emerging biomarker for TNBC.
Abstract
Background: Many prognostic gene signatures have been developed for estrogen receptor positive (ER+) breast cancer (BC); however, most have been solely based on mRNA expression data without ...integrated information on underlying primary drivers such as genomic aberrations. We therefore coupled gene expression and copy number aberration (CNA) in an attempt to improve upon prognostic signatures for ER+ BC.
Methods: mRNA expression based discovery was conducted between 172/59 ER+ BC with low/high Ki67 levels after neoadjuvant aromatase inhibition and significant genes (significance analysis of microarray, q-value less than 0.05) were screened by correlation (Mann-Whitney-Wilcoxon test P less than 0.05) with CNA using Agilent comparative genomic hybridization array. Further interrogation on prognosis of relapse-free survival (RFS) by univariate survival analysis (P less than 0.05) in patients treated with adjuvant endocrine monotherapy from a public data set produced a Copy Number Aberration Driven Endocrine Response (CADER) signature consisting of treatment sensitive/resistant genes. MetaCore (GeneGo Inc) pathway analysis was conducted for enriched pathways. We subsequently applied Nanostring nCounter technology to formalin fixed archival tumor RNA from 620 ER+ adjuvant tamoxifen treated BC (UBC TAM-series) for CADER gene profiling. Patients in multiple independent public data sets and the TAM-series were classified into treatment sensitive defined by up-regulated sensitive gene centroid and down-regulated resistant gene centroid by the median cutoffs, treatment resistant defined with the reverse pattern and indeterminate otherwise. CADER risk stratifications were associated with patient survival outcomes in public cohorts and the TAM-series. The Kaplan-Meier (KM) analysis and Cox models were used for survival analysis. Published PAM50 intrinsic subtypes and subtype based risk of relapse (ROR-S) assignments were used (Nielsen CCR 16:5222, 2010).
Results: A 54-gene CADER signature, 27 resistant/27 sensitive genes, was derived. Pathway analysis indicated that CADER was enriched with sensitive genes of cell survival functions while resistant genes were largely drivers of cell cycle progression. CADER stratifications were significantly prognostic of relapse free survival (RFS) in all public cohorts (log rank test P=0.05 for all) and in the UBC TMA-series (P=0.0001, BC specific survival and RFS). CADER showed an additional value (likelihood ratio test P=0.05) in all cohorts when both standard clinical variables and ROR-S were incorporated in multivariate Cox models. CADER were highly concordant with intrinsic subtypes and ROR-S (p=0.0001) in all data sets. However, CADER may stratify risk within ROR-S medium risk patients (P=0.002 METABRIC; P=0.003 and 0.036 in TAM-series for BC specific survival and RFS).
Conclusions: We have developed a signature that is prognostic of long-term survival in postmenopausal BC, further splits risk within ROR-S medium risk group and identifies some highly resistant BC in presence of ROR-S and clinical variables (see Ellis et. al. abstract for evaluation of a CADER single sample predictor in the MA12 Phase 3 clinical trial).
Citation Format: Jingqin Luo, Li-Wei Chang, Yu Tao, Jeremy Hoog, Samuel Leung, Torsten O Nielsen, Matthew J Ellis. A copy number aberration driven endocrine response gene signature stratifies risk in estrogen receptor positive breast cancer abstract. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-02.
Abstract
Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ~1/3 of patients and acquired resistance in ~1/5 of the remainder. While ...many resistance mechanisms have been explored, therapeutic strategies to overcome resistance in the clinical setting have seen mixed outcomes, and appear most effective in the acquired resistance setting. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges facing breast cancer researchers. In this study we systematically examine the role of DNA damage repair defects in inducing endocrine therapy resistance, a relatively understudied question of recent interest. We use in silico analysis of clinical datasets, in vitro experiments evaluating endocrine therapy resistance in response to DDR dysregulation in multiple breast cancer celllines, and in vivo validation using cellline xenograft and patient-derived xenograft models. We also use gene expression microarrays and RPPA data from cell lines, patient-derived xenografts and primary ER+ breast tumors to uncover therapeutic options that are validated in vitro and in vivo and corroborated by clinical trial data. The results of this study uncover an intriguing link between mismatch repair (MMR) deficiency, specifically of the MutL complex (MLH1/3, PMS1/2), and poor prognosis in ER+ disease. We find a direct role for MutL loss in endocrine therapy resistance in vitro and in vivo by knocking down multiple MutL genes using CRISPR and stable shRNA approaches validated using standard rescue experiments. We identify the underlying mechanism: MutL deficiency in ER+ breast cancer abrogates Chk2-mediated feedback inhibition of CDK4/6 that appears necessary for endocrine therapy responsiveness. Consequently, pharmacological targeting of CDK4/6 in vitro and in vivo significantly inhibits growth of endocrine therapy resistant MutL-deficient ER+ breast cancer cells. These results are corroborated by data from a neoadjuvant clinical trial demonstrating that cell cycle regulation of MutL-mutant tumors tends to be estrogen-independent but sensitive to CDK4/6 inhibitors. The results of this study provide important biological and clinically relevant insights. 1) MMR deficiency is unexpectedly causal to intrinsic endocrine therapy resistance 2) This causal effect appears to be mediated by abrogation of cell cycle checkpoint activation in response to endocrine therapy 3) MMR deficiency in a subset of ER+ tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy resistant tumors. While there are currently no biomarkers to guide the use of CDK4/6 inhibitors for ER+ breast cancer, markers of MMR dysregulation could identify patients in whom CDK4/6 inhibition should be used to prevent disease recurrence.
Citation Format: Svasti Haricharan, Jacob Schmelz, Cheryl Schmidt, Purba Singh, Kimberly R. Holloway, Meenakshi Anurag, Shunqiang Li, Shyam M. Kavuri, Shixia Huang, Dean P. Edwards, Vera Suman, Kelly Hunt, John A. Olson, Jeremy Hoog, Cynthia X. Ma, Matthew N. Bainbridge, Matthew J. Ellis. Mismatch repair defects and endocrine therapy resistance in estrogen receptor positive breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2017-489
The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative ...breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.
The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far ...by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late "transitional" stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)-induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho-guanine nucleotide exchange factors.
Adding fulvestrant to anastrozole (A+F) improved survival in postmenopausal women with advanced estrogen receptor (ER)-positive/ERBB2 (formerly HER2)-negative breast cancer. However, the combination ...has not been tested in early-stage disease.
To determine whether neoadjuvant fulvestrant or A+F increases the rate of pathologic complete response or ypT1-2N0/N1mic/Ki67 2.7% or less residual disease (referred to as endocrine-sensitive disease) over anastrozole alone.
A phase 3 randomized clinical trial assessing differences in clinical and correlative outcomes between each of the fulvestrant-containing arms and the anastrozole arm. Postmenopausal women with clinical stage II to III, ER-rich (Allred score 6-8 or >66%)/ERBB2-negative breast cancer were included. All analyses were based on data frozen on March 2, 2023.
Patients received anastrozole, fulvestrant, or a combination for 6 months preoperatively. Tumor Ki67 was assessed at week 4 and optionally at week 12, and if greater than 10% at either time point, the patient switched to neoadjuvant chemotherapy or immediate surgery.
The primary outcome was the endocrine-sensitive disease rate (ESDR). A secondary outcome was the percentage change in Ki67 after 4 weeks of neoadjuvant endocrine therapy (NET) (week 4 Ki67 suppression).
Between February 2014 and November 2018, 1362 female patients (mean SD age, 65.0 8.2 years) were enrolled. Among the 1298 evaluable patients, ESDRs were 18.7% (95% CI, 15.1%-22.7%), 22.8% (95% CI, 18.9%-27.1%), and 20.5% (95% CI, 16.8%-24.6%) with anastrozole, fulvestrant, and A+F, respectively. Compared to anastrozole, neither fulvestrant-containing regimen significantly improved ESDR or week 4 Ki67 suppression. The rate of week 4 or week 12 Ki67 greater than 10% was 25.1%, 24.2%, and 15.7% with anastrozole, fulvestrant, and A+F, respectively. Pathologic complete response/residual cancer burden class I occurred in 8 of 167 patients and 17 of 167 patients, respectively (15.0%; 95% CI, 9.9%-21.3%), after switching to neoadjuvant chemotherapy due to week 4 or week 12 Ki67 greater than 10%. PAM50 subtyping derived from RNA sequencing of baseline biopsies available for 753 patients (58%) identified 394 luminal A, 304 luminal B, and 55 nonluminal tumors. A+F led to a greater week 4 Ki67 suppression than anastrozole alone in luminal B tumors (median IQR, -90.4% -95.2 to -81.9% vs -76.7% -89.0 to -55.6%; P < .001), but not luminal A tumors. Thirty-six nonluminal tumors (65.5%) had a week 4 or week 12 Ki67 greater than 10%.
In this randomized clinical trial, neither fulvestrant nor A+F significantly improved the 6-month ESDR over anastrozole in ER-rich/ERBB2-negative breast cancer. Aromatase inhibition remains the standard-of-care NET. Differential NET response by PAM50 subtype in exploratory analyses warrants further investigation.
ClinicalTrials.gov Identifier: NCT01953588.
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