Table S1. Overlap of biomarkers measured in OAM4558g; Table S2. Primer/probe information; Table S3. MET diagnostic scoring criteria; Table S4. MET IHC intratumoral heterogeneity; Table S5. Impact of KRAS/EGFR mutation on clinical outcome; Table S6. Biomarker status in EGFR-mutant patients; Table S7. Association of transcript expression with PFS in OAM4558g.

Supplementary Figures S1-4 and Tables S1-6. Supplementary Figure S1. LY6E is overexpressed in a subset of breast cancers Supplementary Figure S2. LY6E transcript expression in normal human tissues is shown. Supplementary Figure S3. Endocytosis of anti-LY6E antibody 9B12 Supplementary Figure S4. LY6E ADC activity in MAXF-1162, a HER2+/ T-DM1 resistant breast cancer model. Supplementary Table S1. Table lists the relative LY6E protein expression detected by IHC Supplementary Table S2. Anti-LY6E antibody 9B12 affinity to human and cynomolgus monkey LY6E. Supplementary Table S3. The correlation of LY6E copy number to anti-LY6E ADC killing in a panel of ovarian cancer cell lines is shown Supplementary Table S4. LY6E and GAPDH specific primer and probe sets used for transcript analysis and LY6E specific siRNAs are listed. Supplementary Table S5. Detailed percent tumor growth inhibition data for xenograft efficacy studies presented in Fig. 6 and in Supplementary Fig. S4B. Supplementary Table S6. Statistical analyses of differences between tumor growth in test groups versus vehicle control group

Figure S1. Characterization of SP44 specificity; Figure S2. Relationship of IHC intensity with FACS; Figure S3. Relationship of IHC intensity with mRNA; Figure S4. Representative IHC images; Figure S5 and S6. MET IHC H-scores; Figure S7. Relationship of IHC clinical score with MET copy number; Figure S8. Kaplan-Meier curves of OS according to MET copy number; Figure S9. Relationship of plasma HGF with MET Dx positivity.

Supplementary Data from VERONICA: Randomized Phase II Study of Fulvestrant and Venetoclax in ER-Positive Metastatic Breast Cancer Post-CDK4/6 Inhibitors – Efficacy, Safety, and Biomarker Results

Abstract Purpose: Despite promising activity in hematopoietic malignancies, efficacy of the B-cell lymphoma 2 (BCL2) inhibitor venetoclax in solid tumors is unknown. We report the prespecified VERONICA primary results, a randomized phase II clinical trial evaluating venetoclax and fulvestrant in estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer, post–cyclin-dependent kinase (CDK) 4/6 inhibitor progression. Patients and Methods: Pre-/postmenopausal females ≥18 years were randomized 1:1 to venetoclax (800 mg orally daily) plus fulvestrant (500 mg intramuscular; cycle 1: days 1 and 15; subsequent 28-day cycles: day 1) or fulvestrant alone. The primary endpoint was clinical benefit rate (CBR); secondary endpoints were progression-free survival (PFS), overall survival, and safety. Exploratory biomarker analyses included BCL2 and BCL extra-large (BCLXL) tumor expression, and PIK3CA circulating tumor DNA mutational status. Results: At primary analysis (cutoff: August 5, 2020; n = 103), venetoclax did not significantly improve CBR [venetoclax plus fulvestrant: 11.8% (n = 6/51; 95% confidence interval (CI), 4.44–23.87); fulvestrant: 13.7% (7/51; 5.70–26.26); risk difference –1.96% (95% CI, –16.86 to 12.94)]. Median PFS was 2.69 months (95% CI, 1.94–3.71) with venetoclax plus fulvestrant versus 1.94 months (1.84–3.55) with fulvestrant (stratified HR, 0.94; 95% CI, 0.61–1.45; P = 0.7853). Overall survival data were not mature. A nonsignificant improvement of CBR and PFS was observed in patients whose tumors had strong BCL2 expression (IHC 3+), a BCL2/BCLXL Histoscore ratio ≥1, or PIK3CA-wild-type status. Conclusions: Our findings do not indicate clinical utility for venetoclax plus fulvestrant in endocrine therapy–resistant, CDK4/6 inhibitor–refractory metastatic breast tumors, but suggest possible increased dependence on BCLXL in this setting.

BACKGROUND: Venetoclax (VEN) is a potent and selective inhibitor of the anti-apoptotic protein, BCL2. Preclinical studies have implicated the BCL2 family members, BCLXL and MCL1, in VEN resistance, and clinical studies in hematological malignancies have demonstrated subgroups with high ratios of BCL2/BCLXL and BCL2/MCL1 have the greatest VEN antitumor activity. The randomized phase 2 VERONICA study (NCT03584009) evaluated VEN in combination with fulvestrant (F) vs F alone in ER-positive, HER2-negative MBC pts who experienced disease recurrence/progression during or after a CDK4/6 inhibitor. Previously reported results from VERONICA (Lindeman et al. ASCO 2021) did not show an improved clinical benefit rate or progression-free survival (PFS) with VEN+F vs F alone. Here we present exploratory biomarker analyses of the expression of BCL2 family members and genomic alterations in circulating tumor DNA (ctDNA) and association with clinical outcomes from VEN+F vs F. METHODS: Tumor specimens were obtained during screening from 103 patients enrolled in the study, and expression levels of BCL2, BCLXL and MCL1 were analyzed by IHC. Baseline plasma-derived ctDNA was evaluated using the FoundationOne® Liquid assay. Expression of BCL2, BCLXL, MCL1 and mutations in ctDNA were correlated with PFS from VEN+F vs F based on the primary analysis (cutoff: Aug 5, 2020). RESULTS: In the overall population, protein levels of BCL2, BCLXL and MCL1 were similar between the VEN+F vs F arms. Patients whose tumors were BCL2 3+ trended towards having the greatest difference in median (m) PFS (3.9 months [mo] in VEN+F vs 1.7 mo in F; hazard ratio [HR] 0.38 [95% CI 0.09, 1.62]) albeit in a small sample size (n=13). Similarly, subgroup analysis suggested a trend for increasing mPFS and improved HR in VEN+F vs F alone in patients with the lowest BCLXL expression. mPFS in patients with a BCL2/BCLXL ratio ≥1 was 3.7 mo for VEN+F vs 1.8 mo for F (HR 0.67 [95% CI 0.3-1.49]) whereas patients with a BCL2/BCLXL ratio <1 had no difference in mPFS between the arms (2.0 mo in both arms, HR 1.21 [95% CI 0.7-2.1]). In the ctDNA-evaluable population, ESR1 (42.6%), TP53 (41.5%) and PIK3CA (35%) were the most prevalent genomic alterations observed and well-controlled between arms. PFS was similar between the ESR1 wildtype (wt) and mutant (mut) subgroups. TP53 mut status was a poor prognostic factor in both treatment arms. The PIK3CA wt subgroup had increased mPFS with VEN+F vs F alone (HR 0.66 [95% 0.38-1.17]) compared to PIK3CA mut (HR 1.59 [95% 0.74-3.34]). Patients with PIK3CA wt and BCL2 high tumors had the largest difference in mPFS between VEN+F (3.7 mo) vs F alone (1.9 mo) (HR 0.58 [95%CI 0.28-1.19]), compared to PIK3CA wt-BCL2 low tumors (2.4 vs 1.9 mo; HR 0.67 [95%CI 0.26-1.72]). CONCLUSION: Our data suggest that a high ratio of BCL2 to BCLXL conferred a trend towards a greater benefit to VEN+F compared to F alone, consistent with other clinical studies evaluating VEN. These analyses highlight the need to profile BCL2 and its family members to identify the VEN-sensitive subgroups, especially in indications where high expression of BCLXL or MCL1 may be observed. The ctDNA profile of VERONICA patients indicate a heavily pretreated patient population. The benefit observed in PIK3CA wt patients from VEN+F suggest increased dependence on BCL2 in this subgroup, while PIK3CA mut tumors likely rely on PI3K/AKT/mTOR survival programs or other BCL2 family members to evade apoptosis. Exploratory biomarker analyses are ongoing to further understand the VERONICA patient population. Citation Format: Geoffrey J. Lindeman, Tharu M. Fernando, Rebecca Bowen, Ching-Wei Chang, Rupal Desai, Kushagra Gupta, Aulde Fléchais, Timothy R. Wilson, Aditya Bardia. Exploratory biomarker analysis in VERONICA, a phase 2 study of venetoclax + fulvestrant versus fulvestrant in patients with estrogen receptor (ER)-positive HER2-negative metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-06.

Abstract Background ~15-20% of primary, invasive breast cancer (BC) overexpresses HER2 and, despite survival improvements, there remains an unmet need for further progress. The antibody-drug conjugate T-DM1 is approved for HER2-positive LA and/or MBC that has previously been treated with trastuzumab and a taxane (separately or in combination), and as adjuvant therapy for HER2-positive early BC, where there is residual invasive disease after neoadjuvant taxane and trastuzumab- or HER2-based treatment. Venetoclax (GDC-0199/ABT-199), an oral, selective small-molecule inhibitor of the antiapoptotic protein BCL-2, is approved for treatment of chronic lymphocytic leukemia, small lymphocytic lymphoma, and acute myeloid leukemia. BCL-2 may play a key role in HER2-positive BC, and venetoclax has shown promising activity in estrogen receptor-positive, BCL-2-positive MBC. We describe VICKI (Venetoclax in Combination with Kadcyla), a Phase Ib/II, randomized, double-blind, placebo-controlled, study of venetoclax plus T-DM1 in previously treated HER2-positive LA/MBC (NCT04298918). Trial design The study comprises a Phase Ib stage (dose escalation and expansion cohorts) and a randomized Phase II stage. Phase II will be initiated following identification of the recommended Phase II dose of venetoclax in Phase Ib (400 mg or 800 mg). Pts will be randomized 1:1 to T-DM1 (intravenous 3.6 mg/kg q3w) plus venetoclax or placebo. Randomization will be stratified per BCL-2 status (BCL-2 high vs. low), visceral disease (Yes vs. No), and HER2 immunohistochemistry (IHC) 3+ status (Yes vs. No). Eligibility Adult pts with HER2-positive (IHC 3+ or IHC 2+/in situ hybridization-positive), previously treated, unresectable, histologically or cytologically confirmed invasive LA/MBC are eligible. Pts will have measurable disease per RECIST v1.1 and an Eastern Cooperative Oncology Group performance status of 0 or 1. Pts in Phase II will have BCL-2 expression status by IHC (≥50% of pts BCL-2 high) and will not have received prior treatment with T-DM1, venetoclax, or anti-HER2 drug conjugates. Aims The Phase II co-primary efficacy endpoints will be objective response rate (ORR) and progression-free survival (PFS) per RECIST v1.1 (both investigator-assessed). Secondary and exploratory efficacy endpoints will include duration of response, overall survival, clinical benefit rate, and patient-reported outcomes. Non-efficacy endpoints will be pharmacokinetics, immunogenicity, biomarkers, and safety. Statistical methods In Phase II, the primary efficacy populations will include all randomized pts according to their assigned treatment arm (intention-to-treat). A point estimate and 95% CI for ORR and the difference in ORR between treatment groups will be calculated using the normal approximation to the binomial distribution. PFS will be defined as time from randomization to the first occurrence of disease progression or death from any cause. Kaplan-Meier methodology will be used to estimate median PFS. An interim analysis is planned when ~56 PFS events have occurred. Primary efficacy analysis will occur when 161 pts have had a PFS event. Cox proportional-hazards models, stratified by the stratification factors, will be used to estimate the hazard ratio with 95% CI. Safety will be analyzed per treatment received in pts who received any study treatment (safety population). Accrual Target accrual is ~226-284 pts at 145 sites globally (Phase Ib dose escalation: 6-24 pts; Phase Ib expansion cohorts: ~20-40 pts; Phase II: 220 pts). Accrual is ongoing. Contact information For more information or to refer a patient, email global-roche-genentech-trials@gene.com or call 1-888-662-6728 (USA only). Citation Format: Geoffrey J Lindeman, Erika Hamilton, Ian Krop, Bora Lim, Shanu Modi, Cristina Saura, Rupal Desai, Bradford J Danner, Tharu M Fernando, Shengchun Kong, Fatema A Legrand, Federico Nasroulah. VICKI: A Phase Ib/II, randomized, placebo-controlled, study of venetoclax plus ado-trastuzumab emtansine (T-DM1) in patients (pts) with previously treated HER2-positive locally advanced (LA) or metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-28-03.

Objective Fenebrutinib (GDC-0853) is a noncovalent, oral, and highly selective inhibitor of Bruton's tyrosine kinase (BTK). The efficacy, safety, and pharmacodynamics of fenebrutinib in systemic lupus erythematosus (SLE) were assessed in this phase II, multicenter, randomized, placebo-controlled study. Methods Patients who had moderately to severely active SLE while receiving background standard therapy were randomized to receive placebo, fenebrutinib 150 mg once daily, or fenebrutinib 200 mg twice daily. Glucocorticoid taper was recommended from weeks 0 to 12 and from weeks 24 to 36. The primary end point was the SLE Responder Index 4 (SRI-4) response at week 48. Results Patients (n = 260) were enrolled from 44 sites in 12 countries, with the majority from Latin America, the US, and Western Europe. The SRI-4 response rates at week 48 were 51% for fenebrutinib 150 mg once daily (P = 0.37 versus placebo), 52% for fenebrutinib 200 mg twice daily (P = 0.34 versus placebo), and 44% for placebo. British Isles Lupus Assessment Group-based Combined Lupus Assessment response rates at week 48 were 53% for fenebrutinib 150 mg once daily (P = 0.086 versus placebo), 42% for fenebrutinib 200 mg twice daily (P = 0.879 versus placebo), and 41% for placebo. Safety results were similar across all arms, although serious adverse events were more frequent with fenebrutinib 200 mg twice daily. By week 48, patients treated with fenebrutinib had reduced levels of a BTK-dependent plasmablast RNA signature, anti-double-stranded DNA autoantibodies, total IgG, and IgM, as well as increased complement C4 levels, all relative to placebo. Conclusion While fenebrutinib had an acceptable safety profile, the primary end point, SRI-4 response, was not met despite evidence of strong pathway inhibition.

1004 Background: For patients (pts) with ER-positive, HER2-negative MBC, CDK4/6 inhibitors + endocrine therapy (ET) is standard first-line treatment, with single-agent ET considered for second-line. Nevertheless, most pts progress. A novel therapeutic target is the antiapoptotic protein BCL2, which is overexpressed in ̃85% of primary ER-positive breast cancers. VEN is a potent, selective BCL2 inhibitor that has shown promising clinical activity in pts with ER-positive and BCL2-positive MBC who have received prior ET. We report the prespecified primary and updated (for overall survival [OS]) analysis of VERONICA (NCT03584009), a phase II study of VEN + F vs F in ER-positive, HER2-negative LA/MBC. Methods: Pts were ≥18-year-old women with ER-positive, HER2-negative LA/MBC, who received ≤2 prior lines of ET and no prior chemotherapy in the LA/MBC setting and experienced disease recurrence/progression during/after CDK4/6 inhibitor therapy (received ≥8 weeks prior). Pts were randomized 1:1 to VEN (oral; 800 mg daily) + F (intramuscular; 500 mg day 1 and 15 of cycle 1; day 1 of subsequent 28-day cycles) or F, and were treated until disease progression, unacceptable toxicity, withdrawal of consent, death, or predefined study end. Pts were stratified by prior lines of therapy in the LA/MBC setting (1 vs 2) and BCL2 status (high vs low). Primary endpoint was clinical benefit rate (CBR; complete response, partial response, and stable disease ≥24 weeks). Secondary endpoints included progression-free survival (PFS) and OS; safety and exploratory subgroup analyses were also conducted. Results: At primary analysis (cutoff: Aug 5, 2020), 103 pts had been randomized (intention-to-treat [ITT] population). Median age was 58.0 and 59.5 years in the VEN + F and F arms, respectively. CBR was similar between arms (VEN + F: 11.8% [n = 6/51; 95% confidence interval (CI) 4.44–23.87]; F: 13.7% [7/51; 5.70–26.26]; risk difference: -1.96% [95% CI -16.86–12.94]). Median PFS was 2.69 months (95% CI 1.94–3.71) in the VEN + F vs 1.94 months (1.84–3.55) in the F arm (stratified hazard ratio: 0.94 [95% CI 0.61–1.45]). Results for CBR and PFS were similar in the BCL2-high and -low subgroups vs the ITT population. More grade 3–4 adverse events (AEs) were observed in the VEN + F vs F arm (n = 13/50 [26%] vs 6/51 [11.8%]). AEs observed with VEN + F were consistent with their individual safety profiles. At updated analysis (cutoff: Oct 22, 2020), OS data were not mature (35.0% event/pt ratio); median OS was 16.99 months in the VEN + F vs not reached in the F arm (stratified hazard ratio: 2.06 [1.04–4.09]). Conclusions: From the primary analysis, VERONICA did not show an improved CBR or PFS with VEN + F, vs F alone, in pts with endocrine- and CDK4/6 inhibitor-refractory LA/MBC. Biomarker analysis is ongoing. Clinical trial information: NCT03584009 .

BACKGROUND Fenebrutinib (GDC-0853, FEN) is a non-covalent, oral, and highly selective inhibitor of Bruton\u0027s tyrosine kinase (BTK). The efficacy, safety, and pharmacodynamics of FEN were assessed in this randomized, placebo-controlled, multi-center phase II study. METHODS Patients with moderate-to-severely active systemic lupus erythematosus on background standard of care therapy were randomized to placebo, FEN 150 mg QD, or FEN 200 mg BID arms. Corticosteroid taper was recommended from weeks 0 to 12 (W0-W12) and W24-W36. The primary endpoint was SRI-4 at W48. RESULTS Patients (N=260) were enrolled from 44 sites in 12 countries, with the majority from Latin America, USA, and Western Europe. The SRI-4 response rates at W48 were 51% (p=0.37, versus placebo) for FEN 150 mg QD, 52% (p=0.34, versus placebo) for FEN 200 mg BID, and 44% for placebo. BICLA response rates at W48 were 53% (p=0.086, versus placebo) for FEN 150 mg QD, 42% (p=0.879, versus placebo) for FEN 200 mg BID, and 41% for placebo. Safety results were similar across all arms, although serious adverse events were more frequent with FEN 200 mg BID. By W48, patients treated with FEN had reduced levels of a BTK-dependent plasmablast RNA signature, anti-dsDNA autoantibodies, total IgG, and IgM, as well as increased complement C4, all relative to placebo. CONCLUSIONS While FEN had an acceptable safety profile, the primary endpoint, SRI-4, was not met despite evidence of strong pathway inhibition.

Background:Bruton’s tyrosine kinase (BTK) plays an essential role in B cell development. BTK acts downstream of the B cell receptor in B cells, and Fc receptor signaling in myeloid cells, pathways thought to be involved in the pathogenesis of RA. Fenebrutinib (FEN) is a non-covalent small molecule inhibitor of BTK with greater than 100-fold selectivity relative to other kinases1.Objectives:To characterize the mechanistic effects of FEN on B and myeloid cell biology and determine the potential of biomarkers to predict response to FEN treatment in patients with RA.Methods:The ANDES study included RA patients (pts) on background methotrexate (MTX) with inadequate response to prior MTX (n=480, Cohort 1, MTX-IR) or anti-TNFs (n=98, Cohort 2, TNF-IR). Cohort 1 pts were randomized to receive PBO, adalimumab (ADA) 40 mg Q2W, or FEN 50 mg QD, 150 mg QD or 200 mg BID. Cohort 2 pts were randomized to receive PBO or FEN 200 mg BID. Clinical efficacy was assessed based on the proportion of pts achieving ACR50 at week 12. Pts for whom samples were available were assessed for levels of rheumatoid factor (RF), total IgM and IgG, CCL4, CXCL13, CRP and IL6.Results:Primary study results are reported separately. Overall, treatment with FEN caused significant reductions in RF (by week 12) and total IgM and IgG (at weeks 4-12) relative to PBO (Table 1). Early and sustained reductions of the B cell chemokine CXCL13 and the myeloid-enriched biomarker CCL4 were observed with FEN or ADA relative to PBO by week 1. CRP levels were significantly reduced with 200 mg BID FEN by week 8, and with ADA by week 2 relative to PBO. By week 12, there was a trend toward lower IL6 levels with FEN treatment relative to PBO, whereas ADA significantly reduced IL6 levels by week 1 relative to PBO in MTX-IR pts. In TNF-IR patients, IL6 was significantly reduced by FEN treatment by week 12 relative to PBO. PK-PD relationships were observed for multiple B and myeloid cell biomarkers. No single biomarker at baseline was associated with clinical response (at 12 weeks) in MTX-IR and TNF-IR pts. However, greater baseline RF titers were associated with increased FEN clinical response in TNF-IR pts.Conclusion:FEN treatment resulted in strong pharmacodynamic effects on multiple biomarkers of B and myeloid cell biology in RA. This included reductions in total IgM and IgG that were not observed with ADA. Conversely, ADA reduced IL6 and CRP faster than FEN, highlighting key mechanistic differences between FEN and ADA. Baseline RF titer was associated with FEN clinical response in the more refractory TNF-IR pts. These data provide mechanistic insights into the efficacy of FEN in RA patients.Reference:[1] Crawford, et al., J Med Chem 2018 61(6) 2227-2245.Disclosure of Interests:Alyssa Morimoto Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Julie Rae Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Leslie Chinn Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Nandhini Ramamoorthi Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Olivia Hwang Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Alexandra Ward Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, D. James Haddon Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Caroline Looney Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Rupal Desai Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Balazs Toth Shareholder of: Stockholder of Genentech/Roche, Employee of: Employee of Genentech/Roche, Katie Tuckwell Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Michael J. Townsend Shareholder of: Stockholder of Genentech/Roche, Employee of: Genentech/Roche

MINT1526A is a monoclonal antibody that blocks the interaction of integrin alpha 5 beta 1 (α5β1) with its extracellular matrix ligands. This phase I study evaluated the safety and pharmacokinetics of MINT1526A with or without bevacizumab in patients with advanced solid tumors.

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.

Abstract Background: The Delta-like 4 (DLL4)-mediated NOTCH signaling pathway is an attractive therapeutic target in cancer. However, chronic blockade of DLL4 signalling has been observed to result in vascular toxicities, such as hepatic sinusoidal dilatation. As the underlying pathogenesis is unclear, the current study was undertaken to interrogate gene expression changes and potential safety biomarkers associated with anti-DLL4 related hepatic sinusoidal dilatation. Methods: Formalin-fixed paraffin-embedded (FFPE) liver sections were derived from male and female cynomolgus monkeys administered FDLL8566 (recombinant humanized anti-DLL4 F(ab’)2 antibody) by intravenous injection once weekly for 8 weeks at dose levels of 0, 5, 15 and 50 mg/kg/week (n = 3/sex/group). RNA was extracted from whole liver sections and laser capture-microdissected (LCM) hepatic regions with or without sinusoidal dilatation. mRNA expression was quantified using a high-throughput RT-qPCR approach, with 96 pre-validated, species-specific TaqMan gene expression assays. A non-parametric statistical test was used to assess differential gene expression between sinusoidal dilatation-affected and non-affected liver tissues. The Benjamini-Hochberg multiple testing comparison error-based False-Discovery-Rate (FDR) method was applied to calculate the adjusted p-values for each probe set. Results: Fourteen candidate genes were differentially expressed between sinusoidal dilatation-affected and non-affected cynomolgus monkey livers with an FDR adjusted p value <0.05. The presence of sinusoidal dilatation could be discriminated based on hierarchical clustering and principal component analysis (PCA). Modulation of expression in genes associated with the VEGF/NOTCH pathway, vascular remodeling, and inflammation were related to anti-DLL4-induced sinusoidal dilatation. Conclusions: This work highlights the involvement of the NOTCH pathway in the maintenance of hepatic sinusoidal homeostasis in the nonhuman primate (NHP). As preclinical toxicities in NHPs have translated to patients for other anti-DLL4 inhibitors, the changes in candidate genes and potential mechanism of toxicity identified in this study are likely to be relevant to humans. The phenotypic characteristics of hepatic sinusoidal dilatation associated with anti-DLL4 resemble the microscopic and molecular features observed in the liver following oxaliplatin treatment of patients (including involvement of angiogenesis), suggesting that similar molecular mechanisms of hepatic toxicity may exist between anti-DLL4 and oxaliplatin. This work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. Citation Format: Monika A. Jarzabek, Rupal Desai, Yuda Zhu, Christina Z. de Zafra, Joe Beyer, Gary Cain, Rajiv Raja, Annette T. Byrne, Priti Hegde, Jacqueline M. Tarrant. Mechanistic insights into the pathogenesis of anti-DLL4-related hepatic sinusoidal dilatation. [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 2096.

Abstract Purpose: DNIB0600A and DMUC5754A are two ADCs that conjugate the anti-mitotic agent MMAE with anti-NaPi2b and anti-MUC16 monoclonal antibodies, respectively. Both ADCs have shown promising anti-tumor activity in patients with platinum resistant ovarian cancer. Here we report biomarker analysis in patient samples collected from these phase 1 studies. The main goal of this study is to evaluate tissue-based biomarkers that can predict response or resistance to these ADCs. We also explored the utility of serum protein biomarkers and circulating tumor cells (CTCs) as potential surrogates for monitoring treatment response to ADCs and disease progression. Methods: Biomarker analysis was done on 55 ovarian cancer patients treated with clinically relevant doses (1.8-3.2mg/kg) from DNIB0600A and DMUC5754A Phase 1 studies. Protein and mRNA expression levels of NaPi2b and MUC16 targets were assessed in archival tumor specimen by immunohistochemistry (IHC) and qRT-PCR respectively. Serum collected at baseline and post-treatment were analyzed by CA125 and HE4 ELISA assays as well as by the OLINK 96-plex PEA protein biomarker panel. CTCs at baseline and post-treatment were analyzed using the Veridex CellSearch System. Results: Target expression in tumor tissues for both NaPi2b and MUC16 measured by IHC and qRT-PCR are concordant. High NaPi2b or MUC16 expression (IHC 2+/3+) was identified in all responders by RECIST criteria (11 from DNIB0600A and 5 from DMUC5754A) for respective target, while no patient from either study with IHC 0 showed RECIST response. In patients treated with DNIB0600A, longitudinal changes in serum CA125 level correlated with RECIST response. Additionally, CTC was detected in 60% of patients at baseline in the DNIB0600A trial, and decreased CTC counts was observed after 1-2 cycles of treatment for two-third of patients. In patients treated with DMUC5754A, circulating CA125 (i.e. extra-cellular domain of MUC16 shed in circulation) is cleared after initial dosing; therefore other ovarian cancer biomarkers including HE4 were assessed. Baseline serum HE4 level correlates well with the tumor burden at pre-treatment in DMUC5754A trial, and showed excellent correlation with RECIST response post-treatment. Conclusions: Target expression in archival tumor tissues is predictive to clinical response to ADCs. CTC enumeration as well as serum HE4 could be used as potential surrogate biomarkers for monitoring treatment response in ovarian cancer. Further validation of these findings is required. Citation Format: Yulei Wang, Ron Firestein, Lisa Ryner, Walter Darbonne, Yinghui Guan, Shan Lu, YJ Choi, Yuanyuan Xiao, Paul Polakis, Becky Suttmann, Rupal Desai, Ling Fu, Ola Saad, Kirsten Achilles Poon, Mitch Denker, Vincent Leveque, Teiko Sumiyoshi, Mark Lackner, David Shames, Eric Humke, Daniel Mayslar. Biomarker evaluation of phase 1 clinical trials of antibody-drug conjugates (ADCs) in platinum resistant ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1441.

Abstract Purpose: Chemotherapies are limited by a narrow therapeutic index resulting in suboptimal exposure of the tumor to the drug and acquired tumor resistance. One approach to overcome this is through antibody–drug conjugates (ADC) that facilitate greater potency via target-specific delivery of highly potent cytotoxic agents. Experimental Design: In this study, we used a bioinformatics approach to identify the lymphocyte antigen 6 complex locus E (LY6E), an IFN-inducible glycosylphosphatidylinositol (GPI)-linked cell membrane protein as a promising ADC target. We developed a monoclonal anti-LY6E antibody and characterized in situ LY6E expression in over 750 cancer specimens and normal tissues. Target-dependent anti-LY6E ADC killing was investigated both in vitro and in vivo using patient-derived xenograft models. Results: Using in silico approaches, we found that LY6E was significantly overexpressed and amplified in a wide array of different human solid tumors. IHC analysis revealed high LY6E protein expression in a number of tumor types, such as breast, lung, gastric, ovarian, pancreatic, kidney and head/neck carcinomas. Characterization of the endocytic pathways for LY6E revealed that the LY6E-specific antibody is internalized into cells leading to lysosomal accumulation. Consistent with this, a LY6E-specific ADC inhibited in vitro cell proliferation and produced durable tumor regression in vivo in clinically relevant LY6E-expressing xenograft models. Conclusions: Our results identify LY6E as a highly promising molecular ADC target for a variety of solid tumor types with current unmet medical need. Clin Cancer Res; 21(14); 3252–62. ©2015 AACR.