RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Phase 1 first-in-human study of dalutrafusp alfa, an anti-CD73-TGF-ß-trap bifunctional antibody, in patients with advanced solid tumors.

BACKGROUND: Cluster of differentiation (CD)73-adenosine and transforming growth factor (TGF)-ß pathways are involved in abrogated antitumor immune responses and can lead to protumor conditions. This Phase 1 study (NCT03954704) evaluated the safety, pharmacokinetics, pharmacodynamics, and efficacy of dalutrafusp alfa (also known as GS-1423 and AGEN1423), a bifunctional, humanized, aglycosylated immunoglobulin G1 kappa antibody that selectively inhibits CD73-adenosine production and neutralizes active TGF-ß signaling in patients with advanced solid tumors. METHODS: Dose escalation started with an accelerated titration followed by a 3+3 design. Patients received dalutrafusp alfa (0.3, 1, 3, 10, 20, 30, or 45 mg/kg) intravenously every 2 weeks (Q2W) up to 1 year or until progressive disease (PD) or unacceptable toxicity. RESULTS: In total, 21/22 patients received at least one dose of dalutrafusp alfa. The median number of dalutrafusp alfa doses administered was 3 (range 1-14). All patients had at least one adverse event (AE), most commonly fatigue (47.6%), nausea (33.3%), diarrhea (28.6%), and vomiting (28.6%). Nine (42.9%) patients had a Grade 3 or 4 AE; two had Grade 5 AEs of pulmonary embolism and PD, both unrelated to dalutrafusp alfa. Target-mediated drug disposition appears to be saturated at dalutrafusp alfa doses above 20 mg/kg. Complete CD73 target occupancy on B cells and CD8+ T cells was observed, and TGF-ß 1/2/3 levels were undetectable at dalutrafusp alfa doses of 20 mg/kg and higher. Free soluble (s)CD73 levels and sCD73 activity increased with dalutrafusp alfa treatment. Seventeen patients reached the first response assessment, with complete response, partial response, stable disease, and PD in 0, 1 (4.8%), 7 (33.3%), and 9 (42.9%) patients, respectively. CONCLUSIONS: Dalutrafusp alfa doses up to 45 mg/kg Q2W were well tolerated in patients with advanced solid tumors. Additional evaluation of dalutrafusp alfa could further elucidate the clinical utility of targeting CD73-adenosine and TGF-ß pathways in oncology.

A phase 1b study of andecaliximab in combination with S-1 plus platinum in Japanese patients with gastric adenocarcinoma.

Andecaliximab (ADX) is a monoclonal antibody that inhibits matrix metalloproteinase 9 (MMP9), an extracellular enzyme involved in matrix remodeling, tumor growth, and metastasis. In preclinical models, MMP9 inhibitors have been shown to enhance the cytotoxic effects of chemotherapeutic agents and to suppress distant metastasis. In this phase Ib, multicenter study, the safety and efficacy of ADX combined with S-1 plus cisplatin (SP) or S-1 plus oxaliplatin (SOX) as a first-line treatment were evaluated in Japanese patients with advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. ADX was administrated at a dose of 800 mg every 2 weeks for the SP cohort and 1200 mg every three weeks for the SOX cohort. As of December 2019, 16 patients were enrolled (six patients in the SP cohort and 10 patients in the SOX cohort). Peripheral sensory neuropathy (69%), anorexia (63%), nausea (56%), and decreased neutrophil counts (44%) were the most common adverse events (AEs). The grade 3 or higher AEs attributed to ADX were stomatitis and abnormal hepatic function (each one patient) in the SP cohort and decreased neutrophil counts (two patients) in the SOX cohort. The objective response rate in 11 patients with measurable target lesions was 73% (8/11), based on the investigator's evaluation. Median progression-free survival was11.9 months (90% confidence interval, 5.6-16.6), and median overall survival was not reached. In conclusion, ADX combined with S-1 plus platinum demonstrated a manageable safety profile and promising clinical activity in the first-line treatment of patients with advanced gastric or GEJ adenocarcinoma.Clinical Trial Registration information: ClinicalTrials.gov Identifier: NCT02862535 (11/08/2016) and protocol ID: GS-US-296-1884.

Safety and tolerability of andecaliximab as monotherapy and in combination with an anti-PD-1 antibody in Japanese patients with gastric or gastroesophageal junction adenocarcinoma: a phase 1b study.

BACKGROUND: Matrix metalloproteinase 9 (MMP9) is implicated in protumorigenic processes. Targeting either stromal or epithelial MMP9 reduces the incidence of metastasis. Andecaliximab is a monoclonal antibody that targets MMP9 with high affinity and selectivity. However, no study has examined whether the inhibition of T-cell programmed death 1 (PD-1) in the presence of andecaliximab increases activated lymphocyte infiltration into the tumor, thereby increasing antitumor activity more than that in anti-PD-1 monotherapy. In this study, we assessed the safety, pharmacokinetics (PK), exploratory biomarkers, and preliminary efficacy of andecaliximab as monotherapy and in combination with nivolumab in Japanese patients with advanced or recurrent gastric or gastroesophageal junction (GEJ) adenocarcinoma. METHODS: This phase 1b study comprised four cohorts enrolling Japanese patients with gastric or GEJ adenocarcinoma. This paper concerns cohorts 1 and 4; cohorts 2 and 3 will be reported subsequently. Cohort 1 enrolled patients with human epidermal growth factor receptor 2 (HER2)-negative tumors (n=8) who received andecaliximab monotherapy (800 mg by intravenous infusion every 2 weeks (Q2W)), and cohort 4 enrolled patients irrespective of their HER2 status (n=10) who received 800 mg of andecaliximab in combination with nivolumab Q2W. Safety, dose-limiting toxicities (DLTs), PK, pharmacodynamics, and biomarkers were assessed in both cohorts. RESULTS: PK of andecaliximab in Japanese patients with gastric or GEJ adenocarcinoma was similar to that reported in non-Japanese patients with advanced solid tumors. Andecaliximab monotherapy and in combination with nivolumab demonstrated no DLTs in cohort 1 and 4, respectively. Toxicities were manageable and well tolerated in both cohorts. The median progression-free survival was 1.4 months (90% CI, 0.5 to 5.4) and 4.6 months (90% CI, 0.9 to not reached) in cohorts 1 and 4, respectively. The objective response rate was 50% (90% CI, 22% to 78%) in cohort 4, and in some patients, the combination therapy was effective regardless of the biomarker status. CONCLUSIONS: The andecaliximab-nivolumab combination demonstrated a manageable safety profile and promising clinical activity in patients with advanced gastric adenocarcinoma.NCT02862535.

Randomized, open-label, phase 2 study of andecaliximab plus nivolumab versus nivolumab alone in advanced gastric cancer identifies biomarkers associated with survival.

BACKGROUND: Matrix metalloproteinase-9 (MMP9) selectively cleaves extracellular matrix proteins contributing to tumor growth and an immunosuppressive microenvironment. This study evaluated andecaliximab (ADX), an inhibitor of MMP9, in combination with nivolumab (NIVO), for the treatment of advanced gastric cancer. METHODS: Phase 2, open-label, randomized multicenter study evaluating the efficacy, safety, and pharmacodynamics of ADX+NIVO versus NIVO in patients with pretreated metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events (AEs). We explored the correlation of efficacy outcomes with biomarkers. RESULTS: 144 patients were randomized; 141 were treated: 81% white, 69% male, median age was 61 years in the ADX+NIVO group and 62 years in the NIVO-alone group. The ORR was 10% (95% CI 4 to 19) in the ADX+NIVO group and 7% (95% CI 2 to 16) in the NIVO-alone group (OR: 1.5 (95% CI 0.4 to 6.1; p=0.8)). There was no response or survival benefit associated with adding ADX. AE rates were comparable in both treatment groups; the most common AEs were fatigue, decreased appetite, nausea, and vomiting. Programmed cell death ligand 1, interferon-γ (IFN), and intratumoral CD8+ cell density were not associated with treatment response or survival. The gene signature most correlated with shorter survival was the epithelial-to-mesenchymal gene signature; high transforming growth factor (TGF)-ß fibrosis score was negatively associated with OS (p=0.036). Gene expression analysis of baseline tumors comparing long-(1+ years) and short-term (<1 year) survivors showed that GRB7 was associated with survival beyond 1 year. Human epidermal growth factor receptor 2 (HER2)-positive disease was associated with significantly longer survival (p=0.0077). Median tumor mutation burden (TMB) was 2.01; patients with TMB ≥median had longer survival (p=0.0025) and improved PFS (p=0.016). Based on a model accounting for TMB, TGF-ß fibrosis, and HER2, TMB was the main driver of survival in this patient population. CONCLUSION: Combination of ADX+NIVO had a favorable safety profile but did not improve efficacy compared with NIVO alone in patients with pretreated metastatic gastric or GEJ adenocarcinoma. HER2 positivity, higher TMB or GRB7, and lower TGF-ß were associated with improved outcomes. TRIAL REGISTRATION NUMBER: NCT02864381 or GS-US-296--2013.

Safety and Efficacy of Andecaliximab (GS-5745) Plus Gemcitabine and Nab-Paclitaxel in Patients with Advanced Pancreatic Adenocarcinoma: Results from a Phase I Study.

BACKGROUND: Matrix metalloproteinase 9 (MMP9) expression in the tumor microenvironment is implicated in multiple protumorigenic processes. Andecaliximab (GS-5745), a monoclonal antibody targeting MMP9 with high affinity and selectivity, was evaluated in combination with gemcitabine and nab-paclitaxel in patients with advanced pancreatic adenocarcinoma. PATIENTS AND METHODS: This phase I study was completed in two parts: part A was a dose-finding, monotherapy phase that enrolled patients with advanced solid tumors, and part B examined andecaliximab in combination with chemotherapy in specific patient cohorts. In the cohort of patients with pancreatic adenocarcinoma (n = 36), andecaliximab 800 mg every 2 weeks was administered in combination with gemcitabine and nab-paclitaxel. Patients were treated until unacceptable toxicity, withdrawal of consent, disease progression, or death. Efficacy, safety, and biomarker assessments were performed. RESULTS: Andecaliximab combined with gemcitabine and nab-paclitaxel appeared to be well tolerated and did not demonstrate any unusual toxicities in patients with pancreatic adenocarcinoma. The most common treatment-emergent adverse events were fatigue (75.0%), alopecia (55.6%), peripheral edema (55.6%), and nausea (50.0%). Median progression-free survival was 7.8 months (90% confidence interval, 6.9-11.0) with an objective response rate of 44.4% and median duration of response of 7.6 months. Maximal andecaliximab target binding, defined as undetectable, andecaliximab-free MMP9 in plasma, was observed. CONCLUSION: Andecaliximab in combination with gemcitabine and nab-paclitaxel demonstrates a favorable safety profile and clinical activity in patients with advanced pancreatic adenocarcinoma. IMPLICATIONS FOR PRACTICE: The combination of andecaliximab, a novel, first-in-class inhibitor of matrix metalloproteinase 9, with gemcitabine and nab-paclitaxel in patients with advanced pancreatic adenocarcinoma provided a median progression-free survival of 7.8 months and objective response rate of 44.4%. The majority of systemic biomarkers related to matrix metalloproteinase 9 activity and immune suppression increased at 2 months, whereas biomarkers related to tumor burden decreased. Although this study demonstrates promising results with andecaliximab plus chemotherapy in patients with advanced pancreatic adenocarcinoma, andecaliximab was not associated with a survival benefit in a phase III study in patients with advanced gastric/gastroesophageal junction carcinoma.

Inhibitory effects of nordihydroguaiaretic acid (NDGA) on the IGF-1 receptor and androgen dependent growth of LAPC-4 prostate cancer cells.

BACKGROUND: Nordihydroguaiaretic acid (NDGA) is an inhibitor of the IGF-1 receptor (IGF-1R) in breast and other cancers, and concomitantly inhibits tumor growth both in cultured cells and animals. The current study evaluates the effect of NDGA on the androgen-stimulated growth of human prostate cancer cells. METHODS: LAPC-4 prostate cancer cells in tissue culture were androgen starved for 3 days, 1 nM dihydrotestosterone (DHT) and other androgens were then added for up to 7 days, and cell proliferation measured. IGF-1R protein expression was measured by Western blot, and IGF-1R mRNA expression by quantitative PCR. IGF-1R receptor kinase activation was measured by ELISA. RESULTS: After 7 days, LAPC-4 growth was doubled by 1 nM DHT. NDGA had a rapid effect to inhibit IGF-1R autophosphorylation induced by IGF-1. DHT increased the expression of IGF-1R protein and mRNA levels. Maximal IGF-1R protein levels were observed 3 days after the addition of androgen. In addition, NDGA, at 10 microM or less, inhibited DHT-induced proliferation in both cells grown in plates and cells grown in soft agar. Androgen receptor (AR) studies by FRET revealed that NDGA had no conformational effects on the AR in response to ligand. CONCLUSIONS: NDGA blocks the DHT-induced growth of LAPC-4 prostate cancer cells by several mechanisms including rapid inhibition of the IGF-1R kinase, and a dose-dependent inhibition of androgen stimulation of IGF-1R expression. Clinical studies of this agent will determine its efficacy in the setting of androgen-dependent prostate cancer.

Nordihydroguaiaretic acid (NDGA), an inhibitor of the HER2 and IGF-1 receptor tyrosine kinases, blocks the growth of HER2-overexpressing human breast cancer cells.

We have reported that nordihydroguaiaretic acid (NDGA) inhibits the tyrosine kinase activities of the IGF-1 receptor (IGF-1R) and the HER2 receptor in breast cancer cells. Herein, we studied the effects of NDGA on the growth of estrogen receptor (ER) positive MCF-7 cells engineered to overexpress HER2 (MCF-7/HER2-18). These cells are an in vitro model of HER2-driven, ER positive, tamoxifen resistant breast cancer. NDGA was equally effective at inhibiting the growth of both parental MCF-7 and MCF-7/HER2-18 cells. Half maximal effects for both cell lines were in the 10-15 microM range. The growth inhibitory effects of NDGA were associated with an S phase arrest in the cell cycle and the induction of apoptosis. NDGA inhibited both IGF-1R and HER2 kinase activities in these breast cancer cells. In contrast, Gefitinib, an epidermal growth factor receptor inhibitor but not an IGF-1R inhibitor, was more effective in MCF-7/HER2-18 cells than in the parental MCF-7 cells and IGF binding protein-3 (IGFBP-3) was more effective against MCF-7 cells compared to MCF-7/HER2-18. MCF-7/HER2-18 cells are known to be resistant to the effects of the estrogen receptor inhibitor, tamoxifen. Interestingly, NDGA not only inhibited the growth of MCF-7/HER2-18 on its own, but it also demonstrated additive growth inhibitory effects when combined with tamoxifen. These studies suggest that NDGA may have therapeutic benefits in HER2-positive, tamoxifen resistant, breast cancers in humans.

Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs.

Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while diminishing its action against other cellular targets. Thus, a set of NDGA analogs (7a-7h) was prepared and examined for inhibitory potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs was based upon a cursory Topliss approach in which one of NDGA's aromatic rings was modified with various substituents. Structural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may serve as a new lead in developing novel therapeutic agents for breast cancer.

Diarylureas are small-molecule inhibitors of insulin-like growth factor I receptor signaling and breast cancer cell growth.

In breast and certain other cancers, receptor tyrosine kinases, including the insulin-like growth factor I receptor (IGF-IR), play an important role in promoting the oncogenic process. The IGF-IR is therefore an important target for developing new anti-breast cancer therapies. An initial screening of a chemical library against the IGF-IR in breast cancer cells identified a diaryl urea compound as a potent inhibitor of IGF-IR signaling. This class of compounds has not been studied as inhibitors of the IGF-IR. We studied the effectiveness of one diaryl urea compound, PQ401, at antagonizing IGF-IR signaling and inhibiting breast cancer cell growth in culture and in vivo. PQ401 inhibited autophosphorylation of the IGF-IR in cultured human MCF-7 cells with an IC50 of 12 micromol/L and autophosphorylation of the isolated kinase domain of the IGF-IR with an IC50 <1 micromol/L. In addition, PQ401 inhibited the growth of cultured breast cancer cells in serum at 10 micromol/L. PQ401 was even more effective at inhibiting IGF-I-stimulated growth of MCF-7 cells (IC50, 6 micromol/L). Treatment of MCF-7 cells with PQ401 was associated with a decrease in IGF-I-mediated signaling through the Akt antiapoptotic pathway. Twenty-four hours of treatment with 15 micromol/L PQ401 induced caspase-mediated apoptosis. In vivo, treatment with PQ401 (i.p. injection thrice a week) reduced the growth rate of MCNeuA cells implanted into mice. These studies indicate that diaryl urea compounds are potential new agents to test in the treatment of breast and other IGF-I-sensitive cancers.

Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-erbB2/HER2/neu receptors and suppresses growth in breast cancer cells.

Nordihydroguaiaretic acid (NDGA) is a phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-cancer activities both in vitro and in vivo. We can now attribute certain of these anti-cancer properties in breast cancer cells to the ability of NDGA to directly inhibit the function of two receptor tyrosine kinases (RTKs), the insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu) receptor. In MCF-7 human breast cancer cells, low micromolar concentrations of NDGA inhibited activation of the IGF-1R, and downstream phosphorylation of both the Akt/PKB serine kinase and the pro-apoptotic protein BAD. In mouse MCNeuA cells, NDGA also inhibited ligand independent phosphorylation of HER2/neu. To study whether this inhibitory effect in cells was due to a direct action on these receptors, we studied the IGF-1-stimulated tyrosine kinase activity of isolated IGF-1R, which was inhibited by NDGA at 10 muM or less. NDGA was also effective at inhibiting autophosphorylation of the isolated HER2/neu receptor at similar concentrations. In addition, NDGA inhibited IGF-1 specific growth of cultured breast cancer cells with an IC50 of approximately 30 muM. NDGA treatment (intraperitoneal injection 3 times per week) also decreased the activity of the IGF-1R and the HER2/neu receptor in MCNeuA cells implanted into mice. This inhibition of RTK activity was associated with decreased growth rates of MCNeuA cells in vivo. These studies indicate that the anti-breast cancer properties of NDGA are related to the inhibition of two important RTKs. Agents of this class may therefore provide new insights into potential therapies for this disease.