Abstract: Clinical trial tissue samples are a valuable resource for biomarker discovery and are frequently collected as Formalin-Fixed, Paraffin-Embedded (FFPE) blocks. FFPE tissues pose challenges for next generation sequencing (NGS)-based expression analysis due to poor yield from highly fragmented RNA. The objective of the current study was to evaluate and compare RNAseq RNAaccess (RR) and Nanostring (NS) platforms for molecular profiling of highly fragmented gastric cancer FFPE tissue. RR (TruSeq RNAaccess, Illumina) and NS (PanCancer immune) were performed according to manufacturer protocol using RNA extracted from sourced FFPE gastric cancer tissues (N=6) with a range of DV200 scores (6-34%) and case-matched fresh-frozen (FF) tissue (N=6). For NS, 200ng and 400ng RNA input levels were tested. Data quality from the 400ng samples proved to be superior, resulting in two more samples passing QC. A final sample size of 4 matched pairs was utilized for cross-platform comparisons. For RR, 100ng RNA was used. Pearson correlation coefficients were examined for the following comparisons: 1) FFPE vs. FF samples from RR, 2) FFPE from NS vs. FF from RR, and 3) FFPE NS vs. FFPE RR. Analyses were performed on log2 transformed expression data, for all available genes (N= 770 common genes from RR and NS) and an 18-gene IFNγ signature (IFNγ). All correlations were plotted against a range of DV200 scores from the FFPE samples in order to evaluate a potential tissue quality cutoff for RR, specifically at DV200 levels below Illumina guideline of 30. All RR data samples yielded & gt;80% exonic rate in uniquely mapped reads. Conversely, samples with DV200 & lt;14 analyzed by NS failed to pass QC due to low fraction of genes detected above background signal. Comparing FFPE to FF tissue from RR, samples with DV200 & gt;10 were highly correlated (r≥0.92), globally or for IFNγ. Comparisons of FF RR to FFPE NS were also well correlated (global median r=0.8; IFNγ median r=0.86). Similarly, expression of FFPE RR was well correlated with FFPE NS (global median r=0.81; IFNγ median r=0.89). Altogether, NS and RR data were highly comparable and minimally impacted by DV200 scores below 30. RR generates gene expression profiles from FFPE tissue that are highly concordant with case-matched FF tissues. This study supports using RR for transcriptional profiling of poor quality gastric cancer FFPE samples. Samples below the Illumina suggested DV200 cutoff of 30 generated good quality RR and NS expression data. IFNγ scores derived from NS and RR from the same tissue were highly concordant. However, NS required higher RNA input level to compensate for low DV200 score and no acceptable expression data was generated on samples below DV200 14. This study will be expanded with data on FFPE samples with DV200 & lt;10 to identify a lower limit of RNA quality for RR. Citation Format: Emon Elboudwarej, Marianna Zavodovskaya, Xi Zhao, Luting Zhuo, Carrie Baker Brachmann, Jinfeng Liu. RNAseq RNAaccess is the preferred method for expression profiling of low quality FFPE gastric cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4528.

Abstract: 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 μmol/L and autophosphorylation of the isolated kinase domain of the IGF-IR with an IC50 & lt;1 μmol/L. In addition, PQ401 inhibited the growth of cultured breast cancer cells in serum at 10 μmol/L. PQ401 was even more effective at inhibiting IGF-I-stimulated growth of MCF-7 cells (IC50, 6 μmol/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 μmol/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. [Mol Cancer Ther 2006;5(4):1079–86]

Abstract: 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.

Abstract: 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 µM 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. J. Cell. Biochem. 103: 624–635, 2008. © 2007 Wiley‐Liss, Inc.

Abstract: 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 µM 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. Prostate 68: 1232–1240, 2008. © 2008 Wiley‐Liss, Inc.

Abstract: The B-cell receptor (BCR) and its downstream effectors have emerged as important therapeutic targets in B-cell malignancies. CC-292 is a novel, potent, covalent, and highly selective inhibitor of Btk (IC50apparent of 0.5 nM, kinact/KI ratio of 7.69 × 104 M-1s-1), that does not appreciably inhibit other kinases involved in BCR signaling (eg, IC50 Lyn kinase, 4401 nM) (Evans et al., J Pharmacol Exp Ther. 2013). Here, we report preclinical characterization and clinical data in CLL from a single-agent phase 1 dose-escalation trial of CC-292 in B-cell malignancies, with a focus on how target engagement and downstream events correlate with clinical activity. Results Pharmacodynamic effects of Btk inhibition by CC-292 can be monitored by occupancy of the Btk catalytic site, Btk autophosphorylation on Y223, and downstream phosphorylation of Plc-γ2 and Erk. We developed a sensitive (10 pg/mL lower limit of quantification) and quantitative assay to measure covalent binding of CC-292 to Btk (Evans et al., J Pharmacol Exp Ther. 2013), as well as Western and novel phos-flow assays to probe downstream signal transduction. These methods showed that CC-292 treatment blocks Btk autophosphorylation and downstream pathway activation in both tumor cells and human peripheral blood mononuclear cells (PBMCs). The extent of CC-292 binding to Btk correlated with its in vitro and in vivo effects. The occupancy assay demonstrated that CC-292 effectively targets Btk in tumor cell lines, PBMCs, spleen, and lymph nodes (LNs) in animal models, and in PBMC and lymph node samples from clinical trial subjects. In rats and non-human primates treated with CC-292, Btk occupancy in spleen and LNs was dose-dependent. Measured occupancy in rat spleen and axillary, mesenteric, and superficial cervical LNs was 94%, 92%, 90%, and 76% respectively, 4 hours (hrs) after a single 30-mg/kg dose. Interim data from the phase 1 CLL trial showed that PBMC Btk was completely occupied in the majority of subjects 4 hrs post-dose with both QD and BID dosing. Twenty-four hrs post-dose at 750 and 1000 mg QD, CC-292 exhibited 83% ± 17% Btk occupancy, whereas with BID dosing at 375 and 500 mg, occupancy was 94% ± 16% at the corresponding time point (12 hrs after the second dose). Thus, while both schedules achieved extensive and sustained Btk occupancy, residual free Btk levels were lower with the BID schedule, offering a rationale for an early trend towards more rapid nodal responses, lymphocytosis, and partial responses on the BID schedule observed to date in the phase 1 study. In the 10 clinical LN biopsies tested to date, no measurable levels of unoccupied Btk have been detected, although Btk protein was present as determined by Western blotting, showing that CC-292 was able to penetrate LNs and inhibit Btk in human subjects as it did in preclinical models. For monitoring downstream signal transduction, we developed reagents and assays including a phos-flow assay based on a novel rabbit monoclonal antibody to detect Btk pY223 levels in PBMC subsets. CC-292 effectively inhibited constitutive and induced phosphorylation of Btk and Plc-γ2 at low nanomolar concentrations. CC-292 also inhibited BCR activation and nurse-like cell–supported survival of CLL cells. Furthermore, CC-292 reduced CLL cell migration and actin polymerization in response to chemokines (CXCL12, CXCL13) and inhibited secretion of the chemokines CCL3 and CCL4 by CLL cells. These chemokines are essential for migration and retention of normal and neoplastic B cells in the marrow and secondary lymphatic tissues. Consistent with this preclinical data, CC-292 treatment resulted in rapid reductions in circulating CCL3 and CCL4 levels. In subjects treated at the 750 mg QD, 1000 mg QD, 375 mg BID, and 500 mg BID dose levels, plasma CCL3 was reduced from 99 ± 16 pg/ml before treatment to 28 ± 5 pg/ml (N = 48, mean ± SEM) at 24 hrs after the first dose, while CCL4 was reduced from 235 ± 59 pg/ml to 74 ± 16 pg/ml (N = 51). Conclusions These data demonstrate that CC-292 achieves significant and durable occupancy of Btk in vitro and in vivo, inhibits Btk-mediated downstream signaling events and chemokine production, and that these preclinical activities have translated into the clinic. Taken together, these results argue that Btk inhibition is necessary and sufficient for clinical activity in CLL. These emerging data support continued development of CC-292 for the treatment of B-cell malignancies. Disclosures: Pierce: Celgene: Employment, Equity Ownership. O'Brien:Genentech: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Consultancy, Research Funding; Talon: Consultancy, Research Funding; Teva/Cephalon: Consultancy. Heise:Celgene: Employment, Equity Ownership. Nacht:Celgene: Employment, Equity Ownership. Aslanian:Celgene: Employment, Equity Ownership. Liu:Celgene: Employment, Equity Ownership. Hong:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Zavodovskaya:Celgene: Employment, Equity Ownership. Marine:Celgene: Employment, Equity Ownership. Barnett:Celgene: Employment, Equity Ownership. Nava-Parada:Celgene: Employment, Equity Ownership. Mei:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Burger:Pharmacyclics: Research Funding; Gilead: Research Funding. Singh:Celgene: Employment, Equity Ownership.

Abstract: 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.

Abstract: 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)-β 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-β 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-β were associated with improved outcomes. Trial registration number NCT02864381 or GS-US-296–-2013.

Abstract: Cluster of differentiation (CD)73-adenosine and transforming growth factor (TGF)-β 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-β 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-β 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-β pathways in oncology.

Abstract: Inhibiting ecto-5’-nucleotidase (CD73) to reduce immunosuppressive adenosine in the tumor microenvironments is an anti-tumor strategy currently explored in clinical trials. Measuring soluble CD73 (sCD73) activity in plasma to evaluate pharmacodynamics of CD73 inhibitors is appealing. Quantifying phosphate in plasma after adding exogenous adenosine monophosphate (AMP) can be used to determine sCD73 activity. 1 Maintaining high plasma concentration to prevent dilution of endogenous sCD73 when quantifying its activity is desirable. High protein concentrations in plasma, however, can affect accurate phosphate quantitation. By precipitating plasma proteins prior to phosphate quantitation, we developed and qualified a method to determine sCD73 activity in 95% plasma. Methods Platelet poor heparinized plasma (PPP), AMP, tissue nonspecific alkaline phosphatase inhibitor (TNAPi), recombinant CD73, malachite green, adenosine 5’- (α, β-methylene) diphosphate (APCP), and recombinant alkaline phosphatase (ALP) were procured commercially. sCD73 concentrations were measured by ELISA and total protein concentration was quantified by BCA. sCD73 activity was measured by combining PPP, TNAPi, and AMP at 37°C. Reactions were terminated with trichloroacetic acid (TCA) at various timepoints to generate a kinetic readout. After protein precipitation, phosphate concentrations were measured by malachite green and enzymatic rates calculated as change in free phosphate concentration per minute. Results Incubating TCA-terminated reaction mixtures at 4°C for ≥ 3 hours reduced protein in supernatants to below lower limits of quantitation and eliminated interference in phosphate detection. Plasma sCD73 activity was dependent on AMP concentrations (Km = 612 μM), proportional to sCD73 in the sample and could be fully inhibited by APCP. 500 μM TNAPi, an inhibitor of non-CD73 AMPase activity, fully blocked 670 IU/L of recombinant human ALP activity. sCD73 activity in PPP from colorectal carcinoma (CRC) patients was higher (p= 0.0028) than in healthy volunteers (HV). sCD73 activity in some individuals with gastric cancer (GC), non-small cell lung cancer (NSCLC), and triple-negative breast cancer (TNBC) were also numerically higher than in healthy volunteers (figure 1a). sCD73 activity was correlated to sCD73 concentrations in these samples (figure 1b). The method was qualified for use in clinical studies (table 1). Abstract 32 Figure 1a sCD73 activity in HV and cancer patient plasma. sCD73 activity in patient plasma from healthy volunteers and solid tumor indications. Abbreviations: HV, healthy volunteer; CRC, colorectal cancer; NSCLC, non-small cell lung cancer; GC, gastric cancer; HNSCC, head and neck squamous cell carcinoma; TNBC, triple-negative breast cancer. Abstract 32 Figure 1b sCD73 activity correlates to sCD73 concentration. Correlation of plasma sCD73 activity and free sCD73 concentrations in patient plasma from healthy volunteers and solid tumor indications. Spearman correlation: r = 0.75, p 〈 0.0001, n = 135. Abbreviations: HV, healthy volunteer; CRC, colorectal cancer; NSCLC, non-small cell lung cancer; GC, gastric cancer; HNSCC, head and neck squamous cell carcinoma; TNBC, triple-negative breast cancer. Abstract 32 Table 1 Plasma sCD73 activity assay qualification limits Plasma sCD73 activity assay characterization and fit-for-purpose qualification. Abbreviations: LOD, limit of detection; LLOQ, lower limit of quantitation; ULOQ, upper limit of quantitation; APCP, adenosine 5’- (α, β-methylene) diphosphate. Conclusions A method to quantify sCD73 activity in 95% plasma to evaluate pharmacodynamics of CD73 inhibitors in clinical samples was developed and qualified. Plasma sCD73 activity was dependent on AMP concentration and inhibited by APCP. Plasma sCD73 activity was significantly elevated in CRC patients and selected patients with GC, NSCLC, and TNBC and was proportional to sCD73 concentration. Reference Morello S, Capone M, Sorrentino C, Giannarelli D, Madonna G, Mallardo D, Grimaldi AM, Pinto A, Ascierto PA. Soluble CD73 as biomarker in patients with metastatic melanoma patients treated with nivolumab. J Transl Med 2017 Dec 4; 15 (1):244. doi: 10.1186/s12967-017-1348-8. PMID: 29202855; PMCID: PMC5716054.