Search

Purpose: Alveolar soft part sarcoma (ASPS) is a rare, highly vascular tumor with few treatment options. We designed a phase II randomized trial to determine the activity and tolerability of single-agent cediranib or sunitinib in patients with advanced metastatic ASPS. Patients and Methods: Patients 16 years of age and older were randomized to receive cediranib (30 mg) or sunitinib (37.5 mg) in 28-day cycles. Patients could cross over to the other treatment arm at disease progression. The primary endpoint was to measure the objective response rate (ORR) for each agent. Median progression-free survival (mPFS) for the two arms was also determined. Results: Twenty-nine of 34 enrolled patients were evaluable for response. One patient on each of the initial two treatment arms had a partial response (ORR: 6.7% and 7.1% for cediranib and sunitinib, respectively). Twenty-four patients had a best response of stable disease (86.7% and 78.6% for cediranib and sunitinib, respectively). There were no significant differences in mPFS for the two treatment arms. Clinical benefit (i.e., objective response or stable disease for a minimum of four or six cycles of therapy) on the first-line tyrosine kinase inhibitor (TKI) therapy did not predict benefit on the second-line TKI. Both drugs were well tolerated. As of August 2021, 1 patient (unevaluable for ORR) remains on study. Conclusions: The study did not meet its endpoints for ORR. Although both TKIs provided clinical benefit, the outcomes may have been attenuated in patients who had progressed ≤6 months before enrollment, potentially accounting for the low response rates. See related commentary by Wilky and Maleddu, p. 1163

This document includes Supplementary Methods, validation data (Figures S1 and S2), complete PD data sets (Figures S3-S7), and Supplementary Tables detailing statistical analyses and assay validation.

Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytic performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kβ inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours postdose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have been in clinical use since 2014 for certain patients with germline

Purpose:The Wee1 kinase inhibitor adavosertib abrogates cell-cycle arrest, leading to cell death. Prior testing of twice-daily adavosertib in patients with advanced solid tumors determined the recommended phase II dose (RPh2D). Here, we report results for once-daily adavosertib.Patients and Methods:A 3 + 3 dose-escalation design was used, with adavosertib given once daily on days 1 to 5 and 8 to 12 in 21-day cycles. Molecular biomarkers of Wee1 activity, including tyrosine 15–phosphorylated Cdk1/2 (pY15-Cdk), were assessed in paired tumor biopsies. Whole-exome sequencing and RNA sequencing of remaining tumor tissue identified potential predictive biomarkers.Results:Among the 42 patients enrolled, the most common toxicities were gastrointestinal and hematologic; dose-limiting toxicities were grade 4 hematologic toxicity and grade 3 fatigue. The once-daily RPh2D was 300 mg. Six patients (14%) had confirmed partial responses: four ovarian, two endometrial. Adavosertib plasma exposures were similar to those from twice-daily dosing. On cycle 1 day 8 (pre-dose), tumor pY15-Cdk levels were higher than baseline in four of eight patients, suggesting target rebound during the day 5 to 8 dosing break. One patient who progressed rapidly had a tumor WEE1 mutation and potentially compensatory PKMYT1 overexpression. Baseline CCNE1 overexpression occurred in both of two responding patients, only one of whom had CCNE1 amplification, and in zero of three nonresponding patients.Conclusions:We determined the once-daily adavosertib RPh2D and observed activity in patients with ovarian or endometrial carcinoma, including two with baseline CCNE1 mRNA overexpression. Future studies will determine whether CCNE1 overexpression is a predictive biomarker for adavosertib.

Supplementary Methods, Supplementary Fig. S1 (Mean adavosertib plasma concentrations for QD vs. BID schedules), Supplementary Fig. S2 (Example PD biomarker immunofluorescence images), Supplementary Fig. S3 (CCNE1 tumor alteration frequency IMPACT data), Supplementary Fig. S4 (Independence of tumor CCNE1 mRNA expression and CCNE1 CNA in TCGA ovarian and endometrial cohorts), Supplementary Table S1 (DLTs), Supplementary Table S2 (Non-DLT dose reductions), Supplementary Table S3 (Responses and baseline genomic characteristics for patients with ovarian carcinoma and prior PARP inhibitor therapy), Supplementary Table S4 (Mean Cycle 1 Day 1 PK parameters), Supplementary Table S5 (Patient tumor mutations identified by WES), Supplementary References

Supplementary Data from Clinical Activity of Single-Agent Cabozantinib (XL184), a Multi-receptor Tyrosine Kinase Inhibitor, in Patients with Refractory Soft-Tissue Sarcomas

Purpose:Soft-tissue sarcomas (STS) are a rare, heterogeneous group of mesenchymal tumors. For decades the mainstay of treatment for advanced, unresectable STS has been palliative chemotherapy. High levels of activated MET receptor have been reported in various sarcoma cell lines, together with elevated vascular endothelial growth factor (VEGF) levels in patients with STS, suggesting that dual targeting of the VEGF and MET pathways with the multi-receptor tyrosine kinase inhibitor cabozantinib would result in clinical benefit in this population.Patients and Methods:We performed an open-label, multi-institution, single-arm phase II trial of single-agent cabozantinib in adult patients with advanced STS and progressive disease after at least 1 standard line of systemic therapy. Patients received 60 mg oral cabozantinib once daily in 28-day cycles, and dual primary endpoints of overall response rate and 6-month progression-free survival (PFS) were assessed. Changes in several circulating biomarkers were assessed as secondary endpoints.Results:Six (11.1%; 95% CI, 4.2%–22.6%) of the 54 evaluable patients enrolled experienced objective responses (all partial responses). Six-month PFS was 49.3% (95% CI, 36.2%–67.3%), with a median time on study of 4 cycles (range, 1–99). The most common grade 3/4 adverse events were hypertension (7.4%) and neutropenia (16.7%). Patients' levels of circulating hepatocyte growth factor (HGF), soluble MET, and VEGF-A generally increased after a cycle of therapy, while soluble VEGFR2 levels decreased, regardless of clinical outcome.Conclusions:Cabozantinib single-agent antitumor activity was observed in patients with selected STS histologic subtypes (alveolar soft-part sarcoma, undifferentiated pleomorphic sarcoma, extraskeletal myxoid chondrosarcoma, and leiomyosarcoma) highlighting the biomolecular diversity of STS.

Purpose: Soft-tissue sarcomas (STS) are a rare, heterogeneous group of mesenchymal tumors. For decades the mainstay of treatment for advanced, unresectable STS has been palliative chemotherapy. High levels of activated MET receptor have been reported in various sarcoma cell lines, together with elevated vascular endothelial growth factor (VEGF) levels in patients with STS, suggesting that dual targeting of the VEGF and MET pathways with the multi-receptor tyrosine kinase inhibitor cabozantinib would result in clinical benefit in this population. Patients and Methods: We performed an open-label, multi-institution, single-arm phase II trial of single-agent cabozantinib in adult patients with advanced STS and progressive disease after at least 1 standard line of systemic therapy. Patients received 60 mg oral cabozantinib once daily in 28-day cycles, and dual primary endpoints of overall response rate and 6-month progression-free survival (PFS) were assessed. Changes in several circulating biomarkers were assessed as secondary endpoints. Results: Six (11.1%; 95% CI, 4.2%–22.6%) of the 54 evaluable patients enrolled experienced objective responses (all partial responses). Six-month PFS was 49.3% (95% CI, 36.2%–67.3%), with a median time on study of 4 cycles (range, 1–99). The most common grade 3/4 adverse events were hypertension (7.4%) and neutropenia (16.7%). Patients' levels of circulating hepatocyte growth factor (HGF), soluble MET, and VEGF-A generally increased after a cycle of therapy, while soluble VEGFR2 levels decreased, regardless of clinical outcome. Conclusions: Cabozantinib single-agent antitumor activity was observed in patients with selected STS histologic subtypes (alveolar soft-part sarcoma, undifferentiated pleomorphic sarcoma, extraskeletal myxoid chondrosarcoma, and leiomyosarcoma) highlighting the biomolecular diversity of STS.

Alveolar soft part sarcoma (ASPS) is a rare, highly vascular tumor with few treatment options. We designed a phase 2 randomized trial to determine the activity and tolerability of single-agent cediranib or sunitinib in patients with advanced metastatic ASPS.Patients 16 years of age and older were randomized to receive cediranib (30 mg) or sunitinib (37.5 mg) in 28-day cycles. Patients could crossover to the other treatment arm at disease progression. The primary endpoint was to measure the objective response rate (ORR) for each agent. Median progression-free survival (mPFS) for the 2 arms was also determined.Results: Twenty-nine of 34 enrolled patients were evaluable for response. One patient on each of the initial 2 treatment arms had a partial response (ORR: 6.7% and 7.1% for cediranib and sunitinib, respectively). Twenty-four patients had a best response of stable disease (86.7% and 78.6% for cediranib and sunitinib, respectively). There were no significant differences in mPFS for the 2 treatment arms. Clinical benefit (i.e., objective response or stable disease for a minimum of 4 or 6 cycles of therapy) on the first-line tyrosine kinase inhibitor (TKI) therapy did not predict benefit on the second-line TKI. Both drugs were well tolerated. As of August 2021, 1 patient (unevaluable for ORR) remains on study.The study did not meet its endpoints for ORR. Although both TKIs provided clinical benefit, the outcomes may have been attenuated in patients who had progressed ≤6 months before enrollment, potentially accounting for the low response rates.

Ras/Raf/MEK/ERK (MAPK) and PI3K/AKT signaling pathways influence several cell functions involved in oncogenesis, making them attractive drug targets. We describe a novel multiplex immunoassay to quantitate isoform-specific phosphorylation of proteins in the PI3K/AKT and MAPK pathways as a tool to assess pharmacodynamic changes. Isoform-specific assays measuring total protein and site-specific phosphorylation levels of ERK1/2, MEK1/2, AKT1/2/3, and rpS6 were developed on the Luminex platform with validated antibody reagents. The multiplex assay demonstrated satisfactory analytic performance. Fit-for-purpose validation was performed with xenograft models treated with selected agents. In PC3 and HCC70 xenograft tumors, the PI3Kβ inhibitor AZD8186 suppressed phosphorylation of AKT1, AKT2, and rpS6 for 4 to 7 hours post single dose, but levels returned to baseline by 24 hours. AKT3 phosphorylation was suppressed in PC3 xenografts at all doses tested, but only at the highest dose in HCC70. The AKT inhibitor MK-2206 reduced AKT1/2/3 phosphorylation in SW620 xenograft tumors 2 to 4 hours postdose, and the MEK inhibitor selumetinib reduced MEK1/2 and ERK1/2 phosphorylation by up to 50% and >90%, respectively. Clinical utility was demonstrated by analyzing biopsies from untreated patients with plexiform neurofibromas enrolled in a clinical trial of selumetinib (NCT02407405). These biopsies showed MEK and ERK phosphorylation levels sufficient for measuring up to 90% inhibition, and low AKT and rpS6 phosphorylation. This validated multiplex immunoassay demonstrates the degree and duration of phosphorylation modulation for three distinct classes of drugs targeting the PI3K/AKT and MAPK pathways.

Background: Differential responses to tamoxifen may be due to inter-patient variability in tamoxifen metabolism into pharmacologically active Z-endoxifen. Z-endoxifen administration was anticipated to bypass these variations, increasing active drug levels, and potentially benefitting patients responding sub-optimally to tamoxifen. Materials and Methods: Patients with treatment-refractory gynecologic malignancies, desmoid tumors, or hormone receptor-positive solid tumors took oral Z-endoxifen daily with a 3+3 phase 1 dose escalation format over 8 dose levels (DLs). Safety, pharmacokinetics/pharmacodynamics, and clinical outcomes were evaluated. Results: Thirty-four of 40 patients were evaluable. No maximum tolerated dose was established. DL8, 360 mg/day, was used for the expansion phase and is higher than doses administered in any previous study; it also yielded higher plasma Z-endoxifen concentrations. Three patients had partial responses and 8 had prolonged stable disease (≥ 6 cycles); 44.4% (8/18) of patients at dose levels 6–8 achieved one of these outcomes. Six patients who progressed after tamoxifen therapy experienced partial response or stable disease for ≥ 6 cycles with Z-endoxifen; one with desmoid tumor remains on study after 62 cycles (nearly 5 years). Conclusions: Evidence of antitumor activity and prolonged stable disease are achieved with Z-endoxifen despite prior tamoxifen therapy, supporting further study of Z-endoxifen, particularly in patients with desmoid tumors.

PURPOSE: We conducted a phase 1 trial of the HSP90 inhibitor onalespib in combination with the CDK inhibitor AT7519, in patients with advanced solid tumors to determine the safety profile and maximally tolerated dose, pharmacokinetics, preliminary antitumor activity, and to assess the pharmacodynamic (PD) effects on HSP70 expression in patient-derived PBMCs and plasma. METHODS: This study followed a 3 + 3 trial design with 1 week of intravenous (IV) onalespib alone, followed by onalespib/AT7519 (IV) on days 1, 4, 8, and 11 of a 21-days cycle. PK and PD samples were collected at baseline, after onalespib alone, and following combination therapy. RESULTS: Twenty-eight patients were treated with the demonstration of downstream target engagement of HSP70 expression in plasma and PBMCs. The maximally tolerated dose was onalespib 80 mg/m(2) IV + AT7519 21 mg/m(2) IV. Most common drug-related adverse events included Grade 1/2 diarrhea (79%), fatigue (54%), mucositis (57%), nausea (46%), and vomiting (50%). Partial responses were seen in a palate adenocarcinoma and Sertoli–Leydig tumor; a colorectal and an endometrial cancer patient both remained on study for ten cycles with stable disease as the best response. There were no clinically relevant PK interactions for either drug. CONCLUSIONS: Combined onalespib and AT7519 is tolerable, though below monotherapy RP2D. Promising preliminary clinical activity was seen. Further benefit may be seen with the incorporation of molecular signature pre-selection. Further biomarker development will require the assessment of the on-target impact on relevant client proteins in tumor tissue.

Background Tumor molecular profiling from patients experiencing exceptional responses to systemic therapy may provide insights into cancer biology and improve treatment tailoring. This pilot study evaluates the feasibility of identifying exceptional responders retrospectively, obtaining pre-exceptional response treatment tumor tissues, and analyzing them with state-of-the-art molecular analysis tools to identify potential molecular explanations for responses. Methods Exceptional response was defined as partial (PR) or complete (CR) response to a systemic treatment with population PR or CR rate less than 10% or an unusually long response (eg, duration >3 times published median). Cases proposed by patients’ clinicians were reviewed by clinical and translational experts. Tumor and normal tissue (if possible) were profiled with whole exome sequencing and, if possible, targeted deep sequencing, RNA sequencing, methylation arrays, and immunohistochemistry. Potential germline mutations were tracked for relevance to disease. Results Cases reflected a variety of tumors and standard and investigational treatments. Of 520 cases, 476 (91.5%) were accepted for further review, and 222 of 476 (46.6%) proposed cases met requirements as exceptional responders. Clinical data were obtained from 168 of 222 cases (75.7%). Tumor was provided from 130 of 168 cases (77.4%). Of 117 of the 130 (90.0%) cases with sufficient nucleic acids, 109 (93.2%) were successfully analyzed; 6 patients had potentially actionable germline mutations. Conclusion Exceptional responses occur with standard and investigational treatment. Retrospective identification of exceptional responders, accessioning, and sequencing of pretreatment archived tissue is feasible. Data from molecular analyses of tumors, particularly when combining results from patients who received similar treatments, may elucidate molecular bases for exceptional responses.

Aims The histone deacetylase inhibitor belinostat has activity in various cancers. Because belinostat is metabolized by the liver, reduced hepatic clearance could lead to excessive drug accumulation and increased toxicity. Safety data in patients with liver dysfunction are needed for this drug to reach its full potential in the clinic. Methods We performed a phase 1 trial to determine the safety, maximum tolerated dose (MTD) and pharmacokinetics of belinostat in patients with advanced cancer and varying degrees of liver dysfunction. Results Seventy-two patients were enrolled and divided into cohorts based on liver function. In patients with mild dysfunction, the MTD was the same as the recommended phase 2 dose (1000 mg/m2 /day). Belinostat was well tolerated in patients with moderate and severe liver dysfunction, although the trial was closed before the MTD in these cohorts could be determined. The mean clearance of belinostat was 661 mL/min/m2 in patients with normal liver function, compared to 542, 505 and 444 mL/min/m2 in patients with mild, moderate and severe hepatic dysfunction. Although this trial was not designed to assess clinical activity, of the 47 patients evaluable for response, 13 patients (28%) experienced stable disease. Conclusion While a statistically significant difference in clearance indicates increased belinostat exposure with worsening liver function, no relationship was observed between belinostat exposure and toxicity. An assessment of belinostat metabolites revealed significant differences in metabolic pathway capability in patients with differing levels of liver dysfunction. Further studies are needed to establish formal dosing guidelines in this patient population.

Purpose: The Wee1 kinase inhibitor adavosertib abrogates cell-cycle arrest, leading to cell death. Prior testing of twice-daily adavosertib in patients with advanced solid tumors determined the recommended phase II dose (RPh2D). Here, we report results for once-daily adavosertib. Patients and Methods: A 3 + 3 dose-escalation design was used, with adavosertib given once daily on days 1 to 5 and 8 to 12 in 21-day cycles. Molecular biomarkers of Wee1 activity, including tyrosine 15–phosphorylated Cdk1/2 (pY15-Cdk), were assessed in paired tumor biopsies. Whole-exome sequencing and RNA sequencing of remaining tumor tissue identified potential predictive biomarkers. Results: Among the 42 patients enrolled, the most common toxicities were gastrointestinal and hematologic; dose-limiting toxicities were grade 4 hematologic toxicity and grade 3 fatigue. The once-daily RPh2D was 300 mg. Six patients (14%) had confirmed partial responses: four ovarian, two endometrial. Adavosertib plasma exposures were similar to those from twice-daily dosing. On cycle 1 day 8 (pre-dose), tumor pY15-Cdk levels were higher than baseline in four of eight patients, suggesting target rebound during the day 5 to 8 dosing break. One patient who progressed rapidly had a tumor WEE1 mutation and potentially compensatory PKMYT1 overexpression. Baseline CCNE1 overexpression occurred in both of two responding patients, only one of whom had CCNE1 amplification, and in zero of three nonresponding patients. Conclusions: We determined the once-daily adavosertib RPh2D and observed activity in patients with ovarian or endometrial carcinoma, including two with baseline CCNE1 mRNA overexpression. Future studies will determine whether CCNE1 overexpression is a predictive biomarker for adavosertib.

PURPOSEThis trial assessed the utility of applying tumor DNA sequencing to treatment selection for patients with advanced, refractory cancer and somatic mutations in one of four signaling pathways by comparing the efficacy of four study regimens that were either matched to the patient's aberrant pathway (experimental arm) or not matched to that pathway (control arm).MATERIALS AND METHODSAdult patients with an actionable mutation of interest were randomly assigned 2:1 to receive either (1) a study regimen identified to target the aberrant pathway found in their tumor (veliparib with temozolomide or adavosertib with carboplatin [DNA repair pathway], everolimus [PI3K pathway], or trametinib [RAS/RAF/MEK pathway]), or (2) one of the same four regimens, but chosen from among those not targeting that pathway.RESULTSAmong 49 patients treated in the experimental arm, the objective response rate was 2% (95% CI, 0% to 10.9%). One of 20 patients (5%) in the experimental trametinib cohort had a partial response. There were no responses in the other cohorts. Although patients and physicians were blinded to the sequencing and random assignment results, a higher pretreatment dropout rate was observed in the control arm (22%) compared with the experimental arm (6%; P = .038), suggesting that some patients may have had prior tumor mutation profiling performed that led to a lack of participation in the control arm.CONCLUSIONFurther investigation, better annotation of predictive biomarkers, and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine. Increasing prevalence of tumor mutation profiling and preference for targeted therapy make it difficult to use a randomized phase II design to evaluate targeted therapy efficacy in an advanced disease setting.

Introduction: Recombinant human interleukin 15 (rhIL-15) has been shown to stimulate the activation and expansion in the number of effector T lymphocytes and natural killer cells in patients. The addition of the immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has shown increased antitumor efficacy in preclinical models, and the triplet is hypothesized to enhance anti-tumor immune response by augmenting effector cell expansion, differentiation, cytotoxic activity, and immune checkpoint inhibition. Results of the lead-in safety arms of rhIL-15/ipilimumab (Ipi) or rhIL-15/nivolumab (N) doublets were previously reported (O’Sullivan et al. AACR. 2019) and we now report results for the dose escalation phase of the triplet combination (NCT03388632). Methods: This phase 1, open-label, 3+3 dose escalation trial enrolled adult patients (pts) with measurable metastatic or refractory solid tumors and ECOG performance status ≤2. Prior therapy with 2 of the 3 study agents was permitted. Pts were treated on three dose levels (DL 1-3) of rhIL-15 (0.5, 1, or 2 mcg/kg/day) administered subcutaneously on days (D)1-8 and D22-29 of a 6-week cycle for the first 4 cycles only. Nivolumab (240mg IV) was administered on D8, 22, 36 and ipilimumab (1mg/kg IV) on D8 of every cycle. Response was assessed by RECIST 1.1. Upon progression, pts could continue for response assessment utilizing iRECIST if clinically stable and without toxicity. Treatment-induced changes in circulating and tumor T cell activation, signaling, and the PD-L1 checkpoint will be assessed with validated pharmacodynamic biomarker assays. Results: A total of 17 pts were enrolled on the triplet dose escalation phase; 15 pts were evaluable for response and 6 pts had received prior treatment with a PD-1/PD-L1 inhibitor. Median time on treatment (MTT) was 12 weeks (range 3-101). A confirmed partial response (PR) was observed in a pt with intrahepatic cholangiocarcinoma (MSI-H) after 3 cycles (total time on treatment 101 weeks). Across all dose levels, 7 pts (46.7%) had stable disease (SD) that prolonged their time on treatment with MTT of 20.4 weeks; 7 pts (46.7%) experienced progressive disease (PD) with MTT of 6.4 weeks. To better profile toxicity, 5 pts were replaced due to incomplete dosing of C1. Dose limiting toxicities at DL 3 (rhIL-15 at 2 mcg/kg/day) were photosensitivity and rash. Main drug-related adverse events included grade 4 lymphopenia (n=1) and grade 3 colitis, hyperthyroidism, hyponatremia, myocarditis, neutropenia, and photosensitivity (n=1, each). No pts discontinued therapy due to toxicity events. No deaths occurred on study. Conclusions: The recommended phase 2 dose (RP2D) of rhIL-15 is 1 mcg/kg/day when administered in combination with N+Ipi. The triplet dose expansion phase with the RP2D is currently accruing and includes biomarker studies in blood and tumor biopsies to assess the tumor microenvironment. This study was funded in part by NCI Contract HHSN261200800001E. *In memorium: Dr. Thomas Waldmann7. Citation Format: Sarah J. Shin, Geraldine O'Sullivan Coyne, Howard Streicher, Naoko Takebe, Ashley Bruns, Elad Sharon, Richard Piekarz, Lamin Juwara, Larry Rubinstein, Ralph Parchment, Kristin Fino, King L. Fung, Katherine Ferry-Galow, Arjun Mittra, Abdul Rafeh Naqash, Kevin Conlon, James H. Doroshow, Alice P. Chen. Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT147.

Background: It is hypothesized that chemotherapy alters the tumor microenvironment (TME) by increasing T cell infiltration and enhancing antigen presentation to T cells, and thereby may enhance immune-mediated cytotoxicity and tumor response to immunotherapy. The goal of this trial (DURVA+) is to evaluate and compare the immune-modulating effects of six different chemotherapy agents with Durvalumab (D), a PD-L1 checkpoint inhibitor. We report here the first analysis of one of the arms of DURVA+: D plus capecitabine (C). Methods: This is a non-randomized phase 2 trial for adult patients (pts) with refractory advanced solid tumors. Pts received C at a dose of 1250 mg/m2 bid on days 1-14 of 21-day cycle. For the first 3 cycles, D is administered every 2 weeks at a dose of 750 mg IV starting on C1D8. From cycle 4 onwards, D was given at a dose of 1125 mg IV every 3 week (day 1 of each 21-day cycle). Tumor biopsies were required at baseline, C1D8 (prior to durvalumab), C3D8, and optional at progression. Radiologic tumor assessment occurs every 4 cycles by RECIST. iRECIST is assessed as a secondary endpoint. Baseline and at least one on-treatment biopsy were obtained for 11 patients, at least 8 of which are evaluable for pharmacodynamic (PD) response (treatment-induced changes in immune cell tumor content and function). Results: As of Nov 10, 2021, 14 pts were enrolled to this arm. Median age 57 (range 31-73 years), 7 males and 7 females, 4 black, 8 white, 2 unknown race. Majority of pts had colorectal carcinoma (ca) (n=8), other include (n=1 each): appendiceal, anal squamous cell ca, leiomyosarcoma, pancreatic ca, breast ca, and nasopharyngeal squamous cell ca. Median lines of prior treatment was 3.5 (1-13), 4 pts previously received ICI. Median number of days on study treatment were 83 days. One pt experience partial response (breast) and five pts experienced SD (3 colon, 1 pancreatic, 1 non-uterine leiomyosarcoma) for a median duration of 136 days as of Jan 3, 2022 (data cutoff); one pt with pancreatic ca experienced a 27% reduction in sum of target lesions. Treatment related adverse events (TrAE) of all grades occurred in 13 pts, serious TrAEs (more than grade 2) occurred in 8 pts and included anemia, pulmonary embolism, hyperbilirubinemia, palmar plantar syndrome, neutropenia, lymphopenia and diarrhea. Immune-mediated AEs included transaminitis/hepatitis (n=4, all grade 1/2), hypothyroidism (n=2, grade 1/2), rash (n=1, grade 1) and colitis (grade 3). PD studies are still ongoing. Conclusion: The combination of durvalumab with capecitabine is feasible with no unexpected adverse events. PD assessment is ongoing to examine the hypothesis that chemotherapy alters tumor micronenvironment (TME) and T cell activation. The study was sponsored by NCI, and was funded in part by NCI Contract HHSN261200800001E. DURVA+ is registered with ClinicalTrials.gov, NCT03907475. Citation Format: Mohamad Adham Salkeni, Geraldine O’Sullivan Coyne, Naoko Takebe, Elad Sharon, Howard Streicher, Jessica Mukherjee, Ashley Bruns, Arjun Mitra, Abdul R. Naqash, Larry Rubinstein, Kristin K. Fino, King L. Fung, Katherine Ferry-Galow, Ralph Parchment, Helen Chen, James Doroshow, Alice Chen. Safety and efficacy of Anti-PD-L1 antibody MEDI4736 (durvalumab) in combination with capecitabine in patients with advanced solid tumors (DURVA+) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT115.

Background: TRC102 binds to abasic sites in DNA, inhibiting the base excision repair (BER) pathway, which is implicated in resistance to alkylating agents. Preclinical xenograft studies in mice demonstrated a synergistic anti-tumor effect when TRC102 was combined with alkylating agents. TRC102 given in combination with temozolomide in a phase 1 trial demonstrated acceptable toxicity and antitumor activity in patients (pts) with colorectal cancer (CRC), non-small cell lung cancer (NSCLC, squamous cell carcinoma) and granulosa cell ovarian cancer (Oncotarget 2020). Pharmacodynamic (PD) analysis revealed treatment-induced induction of nuclear Rad51 signal, indicating DNA damage repair (DDR) response and tumor biopsy data indicated MGMT methylation correlated with tumor response in CRC (Cancer Cell, 2020). Here we report data from a phase II expansion cohort of this combination in pts with NSCLC. Methods: We conducted a single arm open label trial in a pt cohort with advanced NSCLS, who had received at least one line of therapy in the metastatic setting. Pts who received immunotherapy (ICI) and/or molecularly targeted therapy were eligible. Pts received 125 mg TRC102 (100 mg for BSA < 1.6) and 150 mg/m2 TMZ given orally daily for days 1-5 of 28-day cycle (C). Restaging CT scans were carried out after every 2 C and evaluated using RECIST 1.1. Blood from all pts was analyzed for circulating tumor cells (CTCs) at baseline and the beginning of each C. A two-stage design was employed, with at least 2 objective responses needed among the first 16 pts treated on study in order to proceed to the second stage. Results: Sixteen pts with NSCLC (12 adenocarcinoma, 4 squamous) were enrolled between 11/2016 and 6/2021; 12 men and 4 women, median age of 69 yrs (range 33-80), 1 Hispanic, 3 Asian, 2 Black/African American, and 10 Caucasian. Pts received a median of 3 prior lines of therapy (range 1-7), including ICI in all but 1 pt. Twelve pts were evaluable for response. Three pts experienced clinical progression before completing C 2, and 1 pt withdrew consent before completing C 2. Median number of Cs received by pts was 3 (range 1-14); 9 pts experience stable disease (SD) as best response lasting median of 12 wks (range 8-56 wks), and 4 pts experienced prolonged SD lasting greater than 6 Cs (24 wks). Treatment-related grade 3 adverse events reported include lymphopenia (4) and anemia (1). No grade 4 or 5 events were noted. Conclusion: TRC102 in combination with temozolomide was well tolerated with notable efficacy. Although no objective tumor responses were observed in this heavily pretreated population, 9 of 15 pts experienced SD, lasting ≥ 24 wks in 4 pts. Given the limited options for NSCLC treatment following ICI and TKI treatment, this regimen merits further evaluation. Analysis of CTCs is ongoing, as well as additional biomarker evaluation in other treatment cohorts. Funded by NCI Contract No. HHSN261200800001E Citation Format: Mohamad A. Salkeni, Geraldine O’Sullivan Coyne, Jennifer Zlott, Naoko Takebe, Ning Ma, Larry Rubinstein, Richard Piekarz, Jared Foster, Brandon Miller, Ralph Parchment, Abdul R. Naqash, Andrea Voth, James Doroshow, Alice Chen. Phase II trial of TRC102 (methoxyamine HCl) in combination with temozolomide (TMZ) in patients with advanced non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P049.

Background Thymic epithelial tumors are PD-L1–expressing tumors of thymic epithelial origin characterized by varying degrees of lymphocytic infiltration and a predisposition towards development of paraneoplastic autoimmunity. PD-1–targeting antibodies have been evaluated, largely in patients with thymic carcinoma. We sought to evaluate the efficacy and safety of the anti-PD-L1 antibody, avelumab (MSB0010718C), in patients with relapsed, advanced thymic epithelial tumors and conduct correlative immunological studies. Methods Seven patients with thymoma and one patient with thymic carcinoma were enrolled in a phase I, dose-escalation trial of avelumab (MSB0010718C), and treated with avelumab at doses of 10 mg/kg to 20 mg/kg every 2 weeks until disease progression or development of intolerable side effects. Tissue and blood immunological analyses were conducted. Results Two of seven (29%) patients with thymoma had a confirmed Response Evaluation Criteria in Solid Tumors–defined partial response, two (29%) had an unconfirmed partial response and three patients (two thymoma; one thymic carcinoma) had stable disease (43%). Three of four responses were observed after a single dose of avelumab. All responders developed immune-related adverse events that resolved with immunosuppressive therapy. Only one of four patients without a clinical response developed immune-related adverse events. Responders had a higher absolute lymphocyte count, lower frequencies of B cells, regulatory T cells, conventional dendritic cells, and natural killer cells prior to therapy. Conclusion These results demonstrate anti-tumor activity of PD-L1 inhibition in patients with relapsed thymoma accompanied by a high frequency of immune-related adverse events. Pre-treatment immune cell subset populations differ between responders and non-responders. Trial registration ClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013. Electronic supplementary material The online version of this article (10.1186/s40425-019-0723-9) contains supplementary material, which is available to authorized users.

Alveolar Soft Part Sarcoma is one of the less commonly diagnosed soft tissue sarcoma subtypes, an infrequent subtype within the already rare category of human malignancy of sarcoma. In this article we will summarize the histopathological features, natural history and distinct molecular and biological features that have become increasingly appreciated with newer technologies and precision oncology. We will discuss the contemporary management of this disease as well as emerging treatment options.

Background: Ataxia-telangiectasia-related (ATR) protein kinase is central to the repair of damaged DNA through the homologous recombination (HR) pathway. We conducted a phase I trial of cisplatin in combination with the ataxia-telangiectasia-related (ATR) protein kinase inhibitor berzosertib (M6620) and the poly (ADP-ribose) polymerase (PARP) 1/2 inhibitor veliparib, postulating that simultaneous PARP and ATR inhibition impairs DNA repair and induces a “BRCA null”-like phenotype which could potentiate the antitumor activity of cisplatin. We evaluated the safety, maximal tolerated dose (MTD) and preliminary anti-tumor activity of this treatment triplet. The study was conducted through the Experimental Therapeutics Clinical Trials Network (ETCTN). Methods: This open label trial used a 3+3 design. Cisplatin and berzosertib were each administered intravenously on separate, sequential days for two continuous weeks [day (D) 1 and 8; D2 and 9 respectively], together with veliparib orally twice daily during IV therapy and for 24 hours after (D1-3 and 8-10) in 21-day cycles. Prior platinum and PARP inhibitor therapy were permitted. Tumor expression of DNA damage repair (DDR) biomarkers was assessed at the MTD using validated, quantitative immunofluorescence assays. Results: Fifty-three patients (pts) enrolled, 46 pts evaluable for response. The MTD and RP2D dose is cisplatin 40 mg/m2 D1 and D8, berzosertib 210 mg/m2 D2 and D9, and veliparib 200mg BID (D1-3, D8-10). Three patients achieved a confirmed partial response (PR; 5.6%). A further 2 patients had an unconfirmed PR (breast carcinoma, -30%; HGS carcinoma, -36%). Four responders (3 PR and 1 uPR) received prior platinum. Median time on study: 4 cycles (range 1-25). Genomic data was available on four pts achieving a response: ovarian cancer (BRCA-wildtype, ATM unknown; 25 cycles), breast carcinoma (ATM mutation; 14 cycles), SCC of the tongue (Chek2 mutation; 20 cycles), and HGS carcinoma (ATM/BRIP1 mutation; 4 cycles). Twenty-six pts (56.5%) had stable disease. Thirty-five patients (66.0%) required dose reduction in at least 1 agent, most commonly veliparib (25; 47.2%). Most common grade 3 and 4 adverse events were related to myelosuppression: anemia (20; 37.7%), thrombocytopenia (17; 32.1%), leukopenia (13; 24.5%), neutropenia (12; 22.6%), and lymphopenia (11; 20.8%). Ongoing pharmacodynamic analyses of tumor biopsies collected on C1D1 (post-veliparib/cisplatin) and C1D9 (post-veliparib/cisplatin/M6620) have demonstrated a combination-induced increase in RAD51, indicative of replication stress. Conclusions: The BVP combination shows anti-tumor activity in HR-compromised tumors and M6620 further increases the DDR response elicited by combination cisplatin/veliparib. Citation Format: Geraldine O'Sullivan Coyne, Khanh T. Do, Shivaani Kummar, Naoko Takebe, Sarina Piha-Paul, Richard Piekarz, Deborah Wilsker, Brandon Miller, Katherine Ferry-Galow, Ralph Parchment, Lamin Juwara DNP, Mary Jane Ong, Arjun Mittra, James H. Doroshow, Alice P. Chen. Phase I trial of the triplet berzosertib (M6620, VX-970), veliparib and cisplatin (BVP) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT115.

Background: NCI ALMANAC, a systematic in vitro combination drug screen study, identified greater-than-additive activity for the combination of the proteasome inhibitor bortezomib and the nucleoside analog clofarabine. The combination also yielded strong, greater-than-single-agent activity in human tumor xenograft models. Our preclinical findings postulate that the activity of this drug pair may be due to modulation of DNA damage and the intrinsic apoptotic cascade. In an ongoing phase I trial, we evaluate the safety and activity of bortezomib and clofarabine in refractory solid tumors, lymphomas, and myelodysplastic syndromes (MDS). Methods: This is an open-label trial with a 3+3 design, enrolling at least one solid tumor/lymphoma and one MDS patient (pt) at each dose level. Starting doses were clofarabine 1 mg/m2 intravenously (IV) on days (D) 1-5 and bortezomib 0.8 mg/m2 subcutaneously (SC) on D1 and D4 of 21-day cycles. Response was determined per RECIST 1.1, Lugano criteria, and IWG, respectively. Exploratory endpoints include markers of DNA damage and epithelial-to-mesenchymal phenotype transition in blood. Once MTD is declared, a biopsy expansion cohort will enroll to evaluate the mechanism of action for the combination using validated apoptosis multiplex and next-generation sequencing assays. Results: As of October 2020, 22 pts were enrolled with advanced solid tumors (n=18), lymphoma (1), and MDS (3). Median pt age is 62 (range 41-80). Median lines of prior therapy is 3 (range 1-8). Three pts had DLTs in the solid tumor/lymphoma cohort: grade 3 anemia at dose level (DL) 3 (clofarabine 1.5 mg/m2, bortezomib 1 mg/m2); grade 4 neutropenia and grade 4 thrombocytopenia at DL 5 (clofarabine 2 mg/m2, bortezomib 1.3 mg/m2); and grade 4 neutropenia at DL 5. In the solid tumor/lymphoma cohort, grade 3 toxicities possibly attributed to study drugs were anemia (3), lymphopenia (3), thrombocytopenia (1), and frequent premature ventricular contractions (1); grade 4 toxicities were lymphopenia (5), neutropenia without infection (2), and thrombocytopenia (1). The only toxicity possibly attributed to study drugs in the MDS cohort was grade 3 febrile neutropenia in one pt. In the solid tumor/lymphoma cohort, 6 pts achieved a best response of stable disease (SD); 3 patients experienced prolonged SD of ≥ 6 months (1 pt each with colorectal adenocarcinoma, pancreatic adenocarcinoma, and cholangiocarcinoma). In the MDS cohort, 2 pts had SD as a best response (8 months in 1 pt following hypomethylating agent failure). Pharmacodynamic analyses in circulating tumor cells are ongoing. Conclusions: Treatment with bortezomib and clofarabine demonstrated prolonged SD in one pt each with colon adenocarcinoma, pancreatic adenocarcinoma, cholangiocarcinoma, and MDS. Hematological DLTs were seen in 2 pts on DL 5. Currently, patients are enrolling on DL 4 for the solid tumor/lymphoma cohort. Funded by NCI Contract No. HHSN261200800001E. Citation Format: James Q. Nguyen, Geraldine O'Sullivan Coyne, Larry Rubinstein, Shivaani Kummar, Lamin Juwara, Jennifer Zlott, Abdul Rafeh Naqash, Murielle Hogu, Jerry Collins, Apurva Srivastava, Brandon Miller, Ralph E. Parchment, Robert Meehan, Christopher S. Hourigan, Steven Pavletic, James H. Doroshow, Alice P. Chen, Naoko Takebe. Phase I trial of the combination of bortezomib and clofarabine in adults with refractory solid tumors, lymphomas, or myelodysplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT138.

3088 Background: The nucleoside analog Aza-TdC inhibits DNA methyltransferase 1 (DNMT1), which regulates methylation-mediated silencing of tumor suppressor genes. Aza-TdC offers an improvement over traditional DNA methyltransferase inhibitors by virtue of a higher incorporation rate into DNA at lower levels of cytotoxicity. Aza-TdC has also shown improved preclinical antitumor activity compared to other hypomethylating agents in some solid tumor xenograft models. In an ongoing phase I trial, we evaluate the safety and activity of Aza-TdC in patients (pts) with advanced solid tumors. Methods: Adult pts with solid tumors whose disease has progressed on standard therapy or for which there is no standard therapy were treated with Aza-TdC administered orally once a day for 5 days of each week for 2 weeks in 21-day cycles. The study followed Simon accelerated titration design 3, with 1 pt per dose level at 100% dose increments. Accelerated titration continued until 1 pt experienced a dose-limiting toxicity (DLT) or 2 pts experience drug-related grade 2 toxicity at any dose level, after which, a 3 + 3 dose escalation design was used. Intrapatient dose escalation was allowed. Correlative studies included pharmacokinetic assays and pharmacodynamic assays in circulating tumor cells. Results: As of January 2021, a total of 18 pts have been enrolled on study. Median pt age is 61.5 years (range 35-84). Tumor types included colorectal adenocarcinoma (5 pts), sarcoma (3), breast carcinoma (2), and ovarian carcinoma (2). The DLTs at 48 mg were grade 3 rash and grade 3 acute kidney injury in one pt and < 75% of dosing completed in another pt due to grade 3 myelosuppression. Among the 10 pts treated at 32 mg, 1 pt experienced a DLT: grade 4 neutropenia. The maximum tolerated dose (MTD) is 32 mg. Grade 3 or 4 toxicities across all cycles possibly attributable to study drug were leukopenia (6), lymphopenia (6), neutropenia (4), rash (2), febrile neutropenia (1), anemia (1), thrombocytopenia (1), acute kidney injury (1), elevated AST (1), elevated ALT (1), diarrhea (1), and dehydration (1). Of the 14 pts evaluable for response, 11 had a best response of stable disease, and 3 had a best response of progressive disease. Median cycles on study is 4 (range 1-10+). A pt with clear cell ovarian carcinoma has been on study for > 10 cycles with stable disease. Conclusions: At the MTD of 32 mg, Aza-TdC is safe and well tolerated with a toxicity profile similar to currently approved hypomethylating agents. Global DNA methylation profiling, RNAseq, and DNMT immunohistochemical analyses of tumor biopsies are planned for the currently accruing dose expansion cohort. Funded by NCI Contract No. HHSN261200800001E. Clinical trial information: NCT03366116.

11519 Background: ASPS constitutes < 1% of soft tissue sarcomas and frequently presents in adolescents and young adults. There are no approved therapies for ASPS. We are currently evaluating the clinical activity of atezolizumab (atezo), an anti-PD-L1 antibody, in patients (pts) with advanced ASPS. Methods: This is a multicenter, open-label, single-arm phase II study where atezo is administered at a fixed dose of 1200 mg in adults or 15 mg/kg (1200 mg max) in pediatric pts age ≥2 once Q21 days. The primary objective is to determine the objective response rate (ORR) of atezo using RECIST 1.1. Secondary objectives include duration of response and correlating response with the immune effects of atezo in blood and paired tumor biopsies (pre- and post-treatment). Tumor specimens were analyzed with multiplex immunofluorescence immuno-oncology panels to quantify CD8+, PD-1+, and PD-L1+ cells/mm2 in the tumor microenvironment. CD8+ density was calculated as the total number of CD8+ cells divided by the entire area (mm2) of the tumor and invasive margins of the biopsy. Results: As of February 4, 2021, 44 pts have been enrolled. The median age in the study was 31 years (range, 12–70) with equal male: female distribution. 54.5% of pts were Caucasian. Baseline ECOG ≤1 was present in 97.7%. The median time on study was 11.5 months (range, 0.8–40.3 months). At data cutoff, response evaluation was available for 43 pts with an ORR of 37.2% (16/43). One pt experienced a complete response and 15 pts experienced a partial response (PR), of which 14 were confirmed. The median time to confirmed response was 3.5 months (range, 2.1–14.9 months). The median duration of confirmed response was 16.5 months (range, 4.9–38.1 months). Stable disease (SD) was present in 58.1% (25/43). One or more grade 3 adverse events potentially related to atezo were identified in 16.3% (7/43) pts. These include diarrhea, hypothyroidism, transaminitis, anemia, vertigo, extremity pain, myalgia, pneumonitis, rash, and stroke (n = 1 each). No grade 4 or 5 events have been reported. Among 8 cases with evaluable biopsy pairs, both baseline and C3D1 specimens in all cases demonstrated CD8+ T cell infiltration and PD-L1 expression. PD-1 expression was detected at baseline in 5 cases and at C3D1 in 7 cases. In 6 cases (3 SDs and 3 PRs), treatment did not change CD8+ cell density. In the other 2 cases (both PRs), CD8+ density increased > 3x above baseline by C3D1. Analysis of T cell activation using pharmacodynamic response biomarkers, along with whole exome and RNA-seq to evaluate the genomic and transcriptomic landscape of ASPS, are ongoing. Conclusions: Atezo is well tolerated and demonstrates promising single agent activity with durable responses in advanced ASPS. Preliminary tumor biomarker analysis confirms the presence of multiple PD-1/PD-L1 immune checkpoint (IC) components, indicating that advanced ASPS is an ideal candidate for therapeutic IC inhibition. Funded by NCI Contract No HHSN261200800001E. Clinical trial information: NCT03141684.

Introduction: To allow broader application of cancer precision medicine, consideration of how use of molecular abnormalities could enhance the drug development plan and how these findings inform drug combination therapy should begin early. This requires a framework to define molecular ‘actionability,’ and a screening plan for enrichment of clinical trials with patients thought most likely to benefit.Areas covered: Literature was reviewed to illustrate how preclinical studies, including patient derived models and genetically engineered models are used to find initial predictive biomarkers. Co-clinical trials and studies of exceptional responders can postulate which molecular variants predict response. ‘Actionable mutations (or molecular variants)’ are refined during early clinical trials, based initially on preclinical evidence, but gathering additional signals from clinical trials and exceptional responders. Use of biomarkers in early drug development to understand the drug’s interaction with the ...

A small fraction of cancer patients with advanced disease survive significantly longer than patients with clinically comparable tumors. Molecular mechanisms for exceptional responses to therapy have been identified by genomic analysis of tumor biopsies from individual patients. Here, we analyzed tumor biopsies from an unbiased cohort of 111 exceptional responder patients using multiple platforms to profile genetic and epigenetic aberrations as well as the tumor microenvironment. Integrative analysis uncovered plausible mechanisms for the therapeutic response in nearly a quarter of the patients. The mechanisms were assigned to four broad categories���DNA damage response, intracellular signaling, immune engagement, and genetic alterations characteristic of favorable prognosis���with many tumors falling into multiple categories. These analyses revealed synthetic lethal relationships that may be exploited therapeutically and rare genetic lesions that favor therapeutic success, while also providing a wealth of testable hypotheses regarding oncogenic mechanisms that may influence the response to cancer therapy. Profiling multi-platform genomics of 110 cancer patients with an exceptional therapeutic response, Wheeler et al. identify putative molecular mechanisms explaining this survival phenotype in ���23% of cases. Therapeutic success is related to rare molecular features of responding tumors, exploiting synthetic lethality and oncogene addiction.

Background: The oral MEK 1/2 inhibitor selumetinib (50-mg po, BID) has shown remarkable response rates in an ongoing phase-II trial (NCT02407405) in adult patients (pts) with neurofibromatosis type-1 (NF1) (cutaneous neurofibroma (cNF) or inoperable plexiform neurofibromas (PN). In this study, we are evaluating the pharmacodynamics (PD) of selumetinib-induced modulation of key phosphorylation sites on MEK1, MEK2, ERK1, and ERK2 using a new, validated, fit-for-purpose multiplex immunoassay of tumor biopsy extracts that distinguishes specific isoforms. Methods: Core needle tumor biopsies scheduled at baseline and pre-cycle 3 of selumetinib treatment were obtained from patients with a confirmed diagnosis of cNF or PN, snap-frozen in liquid nitrogen within 2 min at point of collection, and stored at -80°C. Additional dermal biopsies were collected (6 of 17 pts) to assess clinical suitability of the proposed PD analysis. Total cell lysates of biopsies were prepared in accordance with the NCI SOP that has been validated to preserve phospho-biomarker integrity (PMID 27001313). Samples were analyzed in a single batch on a Luminex platform, and the ratio of phosphorylated to total protein calculated individually for MEK1, MEK 2, ERK1, and ERK2 and compared by pair-wise analysis. Results: The current PD evaluation resulted from analysis of 24 paired biopsies (17 pts; 6 cNF, 18 PN) representing a range in time interval between previous dose and biopsy procedure (43 min - to 28 hrs; median 7.6, 95% CI 3.25 - 13.98 hrs). Selumetinib treatment decreased the median pERK1 ratio by 59% (95% CI: -30 to -77%; p50% suppression in 12/17 pts. The last dose maintained pERK1/2 suppression (>50% decrease in their ratio values) for >8 hrs in 5 pts and >20 hrs in 2 pts. Conclusions: Our PD study provides the first evidence of engagement and control of selumetinib's molecular target in NF1. Importantly, molecular target response was demonstrable in 14 of 15 patients who responded clinically to this treatment regimen, a finding enabled by a validated high-performance assay for phosphoproteins that are key PD biomarkers of selumetinib mechanism of action and that may be useful in assessing molecular drug action of other members of this drug class. Funded by NCI Contract No HHSN261200800001E. Citation Format: William Herrick, Geraldine O'Sullivan Coyne, Andrea Gross, Eva Dombi, Jeevan Govindharajulu, Casey Kilpatrick, Jennifer Desanto, Amanda Marie Carbonell, Joanne Derdak, Dominique Corinne Pichard, Cecilia Maura Tibery, Bradford J. Wood, Lawrence Doyle, James H. Doroshow, Alice Chen, Ralph E. Parchment, Brigitte C. Widemann, Apurva K. Srivastava. Intra-tumoral pharmacodynamics of selumetinib in serial biopsies from patients with neurofibroma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 805.

Research biopsies collected for evaluation of pharmacodynamic (PD) biomarkers present requirements beyond those obtained for diagnostic purposes. Despite establishing stringent post-collection handling and processing procedures, we have found that many biopsy pairs collected from the early phase trial patient population demonstrate significant tissue heterogeneity and viable tumor content too low to adequately assess biomarker response in the tumor cell population. A retrospective analysis of samples collected from NCI's Developmental Therapeutics Clinic (DTC) from 1/2010-9/2016 revealed an initial success rate for analysis of paired biopsies of only 53%; this was primarily due to a high incidence of biopsies with no or very low tumor content. Based on this initial high failure rate, several clinical and laboratory initiatives were undertaken to identify and mitigate factors which contribute to biopsy unsuitability. After consultation with other translational oncology teams to review best practices, we have published and implemented critical recommendations to improve research biopsy quality at the clinical project team level (PMID: 30285529). In addition to important updates to the clinical trial strategy, several actionable improvements have been identified and implemented at the laboratory level to significantly increase the success rate of research biopsy analyses. Improvements in the biopsy processing approach include use of multiple 18-gauge biopsy cores per timepoint as a standard approach for all PD trials, implementation of methods to prevent biopsy curling during fixation and processing to ensure full longitudinal facing of the tissue during sectioning, and deeper sectioning of the blocks with H&E evaluation of flanking slides by an anatomic pathologist with subsequent release of nonadjacent slides from the most optimal slide range for the primary PD analyses. Development of advanced image analysis methods including the use of β-catenin as a tissue segmentation marker for multiplex immunofluorescence assays has allowed for successful analysis of biopsies with significant heterogeneity and low tumor content. Additionally, defining the minimum cell number required to reach significance for PD marker measurements and capturing additional image fields in low tumor content biopsies to reach the required threshold has led to the implementation of highly robust methods across multiple clinical trials and laboratories. An analysis of biopsies collected from 10/2016-9/2019, after which several clinical and laboratory improvements had been implemented, revealed a significantly improved success rate for paired biopsies of 83%. Funded by NCI Contract No HHSN261200800001E. Citation Format: Katherine V. Ferry-Galow, Deborah Wilsker, Robert J. Kinders, Hala R. Makhlouf, Angie Dull, Tony Navas, Rachel E. Andrews, Geraldine O'Sullivan Coyne, Alice P. Chen, James H. Doroshow, Ralph E. Parchment. Implementation of optimized research biopsy analyses for clinical pharmacodynamic (PD) studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 802.

3624 Background: Adavosertib, a first-in-class Wee1 kinase inhibitor, abrogates G2/M cell cycle arrest causing premature mitosis and DNA replication stress, yielding enhanced DNA damage. Here we report on potential biomarkers of response from tumor genomic analysis in patients (pts) with solid tumors treated with adavosertib. Methods: Adavosertib was administered once daily on days 1-5 and 8-12 of a 21-day cycle. RECIST 1.1 was used to evaluate clinical response. Paired tumor biopsies were obtained for RNASeq gene expression profiling (GEP) and for whole-exome sequencing (WES) to evaluate gene mutation and copy number amplification (CNA). Fold change (FC) was calculated to define gene overexpression. To identify the frequency of CNA and mRNA overexpression for the genomic biomarkers of interest, cBioPortal analysis using TCGA and MSK-IMPACT datasets was performed. Differential GEP analysis of tumor and paired normal tissue was performed using the gene expression profiling interactive analysis (GEPIA) interface (Tang et al. 2017). Results: Out of 35 pts evaluable for response, 6 (17%) had partial response (PR; 4 ovarian carcinoma [OVC], 2 endometrial carcinoma [EC]). The median duration of response was 5.2 months (range 4.0-23.1). Eighteen pts (51.4%) had stable disease. Genomic analysis of tumor biopsies was available for 9 pts; 7 of these pts were evaluable for response, and 3 had PR (2 OVC, 1 EC). WES revealed TP53 mutations in 6 pts (66.6%; 3 pts with PR, 2 with progressive disease,1 not evaluable). On WES, tumor Cyclin E1 ( CCNE1) CNA was present in 1 of 3 PR pts while tumors from all 3 PR samples showed relatively high CCNE1 expression by RNAseq (FC = 4.07). In the MSK-IMPACT 2017 dataset, CCNE1 CNA was identified in 1.8% of pts (194 of 10336); of which, OVC (10.3%) and EC (8.7%) had the highest incidence of CCNE1 CNAs. In separate tumor-specific (OVC, EC) TCGA datasets having CCNE1 overexpression and/or CNA, overlap in CCNE1 overexpression with CCNE1 CNA was 35.5% (OVC) and 25.2% (EC). Compared to normal ovarian/ endometrial tissues, GEPIA analysis revealed significantly higher CCNE1 mRNA expression in OVC (FC = 3.5) and EC (FC = 3.8). Conclusions: CCNE1alterations (overexpression and/or CNA) tend to be enriched in OVC and EC with a limited fraction showing both overexpression and CNA. Tumor genomic analysis of additional OVC and EC pts treated with adavosertib is required to determine whether CCNE1 mRNA overexpression, regardless of CCNE1 CNA, is a potential biomarker of response to this drug in these tumor types. Funded by NCI contract No. HHSN261200800001E. Clinical trial information: NCT01748825 .

3612 Background: NF1-related PN are locally invasive tumors characterized by increased activation of the RAS pathway causing significant morbidity including disfigurement, pain and functional limitations. Selumetinib has received breakthrough designation for NF1 PN based on phase I / II trials in children. We present results for the ongoing phase II study of selumetinib in adults with NF1 PN, which includes pharmacodynamic (PD) evaluation of serial tumor biopsies as well as functional/patient-reported outcomes (PROs). Methods: Open-label Simon 2-stage design. Eligibility: NF1 patients (pts) ≥18 years old with inoperable/symptomatic/progressive PN. First 2 pts received selumetinib 75 mg BID; subsequent pts received selumetinib 50 mg BID. Primary objective: response rate by volumetric MRI analysis (partial response [PR]; ≥20% volume decrease). Secondary objectives: PD studies on pre/on-treatment biopsies of PN and cutaneous neurofibromas, assessment of clinical benefit using PROs (Numeric Rating Scale-11, Pain Interference Index), and PN-specific functional assessments. Validated, fit-for-purpose, isozyme-specific measurements of pERK/ERK/pMEK/MEK performed using SOPs designed for labile phosphoproteins (PMID 27001313). Results: As of February 2020, 27 pts have enrolled. Outcomes are reported for 23 pts (74% male; median age 33 years, range 18-60). Most common PN-related morbidity: pain (19 pts). Sixteen pts achieved PR (69%), with 13/16 confirmed; no disease progression. Time to response: 11 months (range 5-25); median change in PN volume at best response: -22% (range -41% to +5.5%); median duration of treatment: 28 months (range 2-50). Selumetinib suppressed tumor pERK1,2/ERK1,2 but not pMEK1,2/MEK1,2 ratios from 1-10 hours following oral dosing (one t½). Pt-reported target tumor pain intensity and pain interference scores significantly improved (both p < 0.03). Pts 1 and 2 were dose-reduced due to grade 3 intolerable rash (n = 2) and pain (n = 1). Grade ≥3 drug-related toxicities on 50 mg (21 pts) include transaminitis (5 pts), rash (1 pt) and pancreatic enzyme elevation (1 pt). Two pts were dose reduced (rash = 1 pt, transaminitis = 1 pt). Two pts discontinued by choice, 2 pts withdrawn by PI (best interest of patient), and 1 pt each removed for transaminitis, surgical resection, serious concurrent medical illness, and non-compliance. Conclusions: Selumetinib shrinks the majority of adult PN and results in molecular target suppression and clinical benefit. Clinical trial information: NCT02407405 .

Epithelial ovarian cancer continues to have a very high mortality rate, partly due to the challenges of early stage diagnosis as well as the low cure rate of standard treatments to date. This chapter will concentrate on the recent updates in the molecular classification of epithelial ovarian cancer, approval of novel targeted agents, and emerging therapeutic modalities.

The 90-kDa heat shock protein HSP90 is a member of a highly evolutionarily conserved class of molecular chaperone proteins indispensable for the development of cancer; when activated by cellular stress, HSP90 stabilizes oncogenic substrate “client” proteins involved in cellular processes that promote tumorigenesis. HSP90 inhibition attenuates this stabilization of aberrant client proteins in tumor cells, allowing for simultaneous targeting of multiple pathways involved in cancer cell survival. HSP90 inhibitors have been assessed as potential oncologic therapies in several preclinical and clinical studies. Although preclinically promising results have been measured, these results have not translated yet into major clinical efficacy. Combinations of HSP90 inhibitors with approved and investigational oncology drugs may represent further opportunities for the use of these agents in patients with cancer. This chapter reviews some of the important early clinical milestones observed in studies of first- and second-generation HSP90 inhibitors used as single agents and in combination. In the conclusion, possible reasons for the lack of therapeutic benefit in clinical studies are considered.

Inflammation is an established process in colorectal cancer development and a hallmark of progression, and pro-inflammatory cytokines have been implicated in the morbidity and functional compromise associated with malignancy. MABp1, described as a first-in-class true human antibody against interleukin-1α, has undergone clinical trial evaluation in a number of indications, recently completing late phase clinical trial testing under Fast Track designation for cancer anorexia-cachexia syndrome in colorectal cancer patients. To date, MABp1 has been evaluated as a novel therapeutic strategy to ameliorate phenotypic factors associated with poor prognosis in colorectal cancer patients. Areas covered: In this review, the authors discuss the clinical trial data available to date for this antibody in colorectal cancer, including novel clinical trial endpoints utilized to evaluate sarcopenia and inflammation, as well as the proposed role of interleukin-1α antagonism in leading to improved patient outcomes. Expert opinion: There is a multitude of antibodies in therapeutic development in oncology, and MABp1 is a novel class of antibody which has been safely tolerated to date. Clinical studies of this agent suggest a significant improvement in lean body mass, though additional results evaluating the impact of targeting inflammation as a strategy to delay disease progression in this population are awaited.

"Precision" trials, using rationally incorporated biomarker targets and molecularly selective anticancer agents, have become of great interest to both patients and their physicians. In the endeavor to test the cornerstone premise of precision oncotherapy, that is, determining if modulating a specific molecular aberration in a patient's tumor with a correspondingly specific therapeutic agent improves clinical outcomes, the design of clinical trials with embedded genomic characterization platforms which guide therapy are an increasing challenge. The National Cancer Institute Precision Medicine Initiative is an unprecedented large interdisciplinary collaborative effort to conceptualize and test the feasibility of trials incorporating sequencing platforms and large-scale bioinformatics processing that are not currently uniformly available to patients. National Cancer Institute-Molecular Profiling-based Assignment of Cancer Therapy and National Cancer Institute-Molecular Analysis for Therapy Choice are 2 genomic to phenotypic trials under this National Cancer Institute initiative, where treatment is selected according to predetermined genetic alterations detected using next-generation sequencing technology across a broad range of tumor types. In this article, we discuss the objectives and trial designs that have enabled the public-private partnerships required to complete the scale of both trials, as well as interim trial updates and strategic considerations that have driven data analysis and targeted therapy assignment, with the intent of elucidating further the benefits of this treatment approach for patients.

The past decade has seen the development and widespread use of tyrosine kinase inhibitors (TKIs) targeting a mutated EGFR (mEGFR) for the treatment of metastatic NSCLC. We discuss the main properties of the TKIs currently recommended for the treatment of mEGFR NSCLC: gefitinib, erlotinib and afatinib.The mechanism of action, pharmacodynamics and pharmacokinetics of these drugs, with emphasis on the historical context of their preclinical and clinical development, will be covered, including potential resistance mechanisms to these first-generation TKIs that has driven the trial design for second and third generations of EGFR inhibitors. Six Phase III clinical trials comparing these three TKIs with cisplatin-based chemotherapy upfront for mEGFR NSCLC provide the basis for the comparative safety and toxicity analysis between these agents. Class-related toxicity of these EGFR inhibitors, including life-threatening effects, will be discussed.Toxicity and safety analysis from the Phase III trials of these agents in mEGFR populations suggests that afatinib has more frequent and severe side effects. Given that an efficacy advantage has not yet been demonstrated for afatinib over erlotinib and gefitinib, the consistent class toxicity profile of these agents means that gefitinib and erlotinib are a safer first-line treatment recommendation.

Monoclonal antibodies and small molecules targeting the VEGF pathway are part of the arsenal to treat malignant tumors. Antiangiogenesis therapies has been studied in breast cancer with partial success, reflected by the approval of bevacizumab in Europe but not in United States, for metastatic breast cancer (mBC). Ramucirumab is a mAb against VEGFR-2 interfering with the normal activation of this receptor by its natural ligand VEGF.This article will review the preclinical data available to date for ramucirumab, as well as survey the main clinical trials of antiangiogenic agents reported in breast cancer, focusing on Phase III clinical trials. It will also review the clinical trial data for ramucirumab in mBC, including the design of the Phase II trials, and report on the preliminary results of the TRIO-012 trial. This trial did not meet its primary end point in progression-free survival and has to be considered as a negative trial.Despite preliminary positive data with ramucirumab in other metastatic solid tumors reported to date, the results of TRIO-012 discourage pursuing more efforts with ramucirumab in mBC unless predictive and reproducible biomarkers can be established to select those patients who are most likely to benefit from it.

Background: We initiated the systematic NCI-ALMANAC in vitro combination drug screen of FDA-approved anticancer drugs in the NCI-60 tumor cell line panel to explore novel and effective drug combinations (Holbeck et al., Cancer Res 2017). In this screen and in preclinical xenograft models, the BCR-ABL kinase inhibitor nilotinib demonstrated greater-than-additive activity in combination with the anti-tubulin agent paclitaxel. Additionally, recent preclinical findings suggest a reduced incidence of peripheral neuropathy for this combination relative to paclitaxel monotherapy (Leblanc et al., J Clin Invest 2018). We are performing a phase 1 study to establish the safety, tolerability, and maximum tolerated dose (MTD) of this combination in patients (pts) with advanced solid tumors, and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to better understand the mechanism of synergy. Materials and Methods: Nilotinib was given orally twice-daily (BID), while paclitaxel was administered intravenously on days 1, 8, and 15 of each 28-day cycle. A 1- or 2-day paclitaxel-only run-in during the first cycle enabled comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Dose-limiting toxicities (DLTs) were assessed during cycle 1, and response was evaluated by CT per RECIST 1.1. Results: Thirty pts have been enrolled to date. The MTD was 300 mg nilotinib BID and 80 mg/m2 paclitaxel given on days 1, 8, and 15; DLTs were grade 4 rash and grade 3 myelosuppression. Only 2 pts (7%) have experienced grade 2 peripheral neuropathy, no grade ≥3 neuropathy events occurred. Of the 24 pts assessable for response to date, there are 4 confirmed partial responses (PRs; 17%, 1 pt with endometrial cancer, 1pt with anal cancer and 2 pts with granulosa cell of the ovary) and 12 pts with a best response of stable disease (SD; 50%), including 6 pts with SD for ≥ 8 cycles. Mean time on study to date is 8.7 months (range: 2 – 41 months). Three of 4 responding pts had undergone prior paclitaxel-based therapy and experienced a best response of only SD (1 pt) or progressive disease (2 pts) to carboplatin/paclitaxel or paclitaxel monotherapy, respectively. PK data demonstrate dose-dependent plasma paclitaxel concentrations (for 60 vs. 80 mg/m2 paclitaxel), and no substantial effects of nilotinib on paclitaxel plasma concentrations. Conclusions: The combination of nilotinib and paclitaxel is well-tolerated. Consistent with recent preclinical findings, no grade ≥ 3 peripheral neuropathy has been observed, as compared to rates of 8-30% in prior studies of 80-100 mg/m2 weekly paclitaxel monotherapy in pts with metastatic breast cancer (Rivera and Cianfrocca, Cancer Chemother Pharmacol 2015). PK data indicate dose-dependent plasma paclitaxel concentrations, with no substantial nilotinib-induced changes in plasma paclitaxel levels. Preliminary response data are promising, especially considering the PRs observed in pts who had previously been unresponsive to paclitaxel therapy. Accrual to the expansion cohort and PD analyses of circulating tumor cells and tumor biopsy specimens are ongoing. Citation Format: Geraldine O'Sullivan Coyne, Shivaani Kummar, Jennifer Zlott, Lamin Juwara, Naoko Takebe, Murielle Hogu, Larry Anderson, Jerry Collins, Richard Piekarz, Howard Streicher, Elad Sharon, Arjun Mittra, Sabrina Khan, Brandon Miller, Tony Navas, Apurva Srivastava, Ralph Parchment, Laurence Rubinstein, James H Doroshow, Alice P Chen. Tolerability and antitumor activity of paclitaxel is improved by the addition of nilotinib in patients with refractory solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C008. doi:10.1158/1535-7163.TARG-19-C008

Background: The PI3K pathway is frequently dysregulated in cancer, promoting tumorigenesis by facilitating aberrant cell growth, blocking apoptotic signaling, and suppressing immune surveillance in the tumor microenvironment (TME). The TME contains regulatory immune cells such as FOXP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells, M2 macrophages, and N2 neutrophils, maintaining an immunologically isolating sheath. PI3K inhibition has been shown to slow tumor growth by inducing apoptosis and sensitizing tumors to T cell-mediated cytotoxicity. Copanlisib is a potent PI3K inhibitor, and combination with the PD-1 checkpoint inhibitor nivolumab is hypothesized to result in robust anti-tumor T cell responses. Materials and Methods: Open label phase I trial of copanlisib and nivolumab following a 3+3 design with an expansion phase at the maximally tolerated dose (MTD). Flat-dose nivolumab is administered intravenously (IV) on day 1 while copanlisib is administered IV weekly on days 1, 8 and 15 of a 28-day cycle; there is a 2-week copanlisib run-in during cycle 1 (expansion phase) to enable assessment of pharmacodynamic (PD) endpoints. Prior checkpoint inhibitor therapy is not permitted. Dose-limiting toxicities (DLTs) were assessed during cycle 1, and responses are assessed by CT using RECIST 1.1 and iRECIST. Exploratory PD objectives include assessments on blood and tissue samples collected at baseline and post copanlisib -/+ nivolumab treatment; these studies will evaluate immune cell populations, PD-1/PD-L1 levels, markers of T cell activation/inhibition (Zap70 pY493 and SHP2 pY542), and apoptosis biomarkers using validated immunofluorescence and flow cytometry assays. Results: As of July 2019, 16 pts have been enrolled with 6 pts in the expansion phase; 14 pts are evaluable for toxicity. The MTD for the combination is copanlisib 60 mg days 1, 8 and 15 plus nivolumab 480 mg day 1 (recommended doses for each individual agent, 28-day cycles); no DLTs were identified. Median age on study is 62 (range 38-77). One pt (rectal cancer) has experienced a confirmed partial response (PR), and 4 pts have achieved stable disease (SD; 1 pt remains on study ≥10 months). Five pts are too early to assess for response. Tissue for molecular analysis and/or genomic sequencing has been collected for all patients. Adverse events include: grade 3 hypertension (1 pt, requiring dose reduction) and infusion-related reaction (hypertension, 1pt); grade 1/2 events include hypertension (7 pts), hyperglycemia (5 pts), nausea (5 pts) and infusion-related reaction (hypertension, 4 pts). Preliminary PD assessments show increases in the number of CD8+ T cells in peripheral blood samples after treatment, with changes in phospho-biomarkers of T cell activation (Zap70-pY493 and SHP-pY542). Conclusions: The combination is tolerable using the approved doses of each individual agent and shows treatment-induced immune PD changes as well as early anti-tumor activity. Enrollment at the expansion stage is ongoing (NCT03502733). Funded by NCI Contract No. HHSN261200800001E. Citation Format: Geraldine O'Sullivan Coyne, Timothy A Yap, Nancy Moore, Arjun Mittra, Naoko Takebe, Lamin Juwara, Sabrina Khan, Ecaterina Ileana Dumbrava, Howard Streicher, Richard Piekarz, Elad Sharon, Funda Meric-Bernstam, Ralph Parchment, Tony Navas, King Leung Fung, James Mitchell, Kristin Fino, Sandra Montez, Austin Doyle, Laurence Rubinstein, James H Doroshow, Alice P Chen. Phase IB combination study of copanlisib and nivolumab in advanced solid tumors and lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B105. doi:10.1158/1535-7163.TARG-19-B105

Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018

Background: The base excisional repair (BER) pathway plays an important role in the development of resistance to alkylating agents. TRC102 (methoxyamine HCL) inhibits BER by binding apurinic/apyrimidinic sites, which increases temozolomide (TMZ)-induced DNA strand breaks and apoptosis. We conducted a multihistology phase I/II trial of TRC102 in combination with TMZ in patients (pts) with refractory solid tumors (NCT01851369); here we report the clinical activity of this combination in the phase II refractory colorectal carcinoma (CRC) cohort. The phase I portion of the study demonstrated anemia to be the major dose limiting toxicity of this combination; the recommended phase II doses (RP2D) for the combination were: 125 mg TRC 102 + 150 mg/m2 TMZ (PO, D1-5 of 28-day cycles). The expanded phase II trial utilized a 3-arm Simon 2-stage design based on objective responses documented during the phase I portion of the study (CRC, NSCLC, and granulosa cell ovarian cancer). Methods: Phase II pts were treated at the RP2D, except that pts with BSA < 1.6 m2 received 100 mg of TRC102. Eligibility for the CRC cohort included pts with recurrent, metastatic, histologically confirmed colorectal adenocarcinoma after ≥ 2 lines of therapy, adequate organ function, and an ECOG performance status (PS) ≤ 1. Overall response rate (ORR) was defined as a partial response (PR) or a complete response (CR), per RECIST 1.1 criteria. Results: During the phase I trial, one pt with KRAS mutant CRC experienced a sustained PR of 21 cycles (maximum 70% decrease by RECIST). This pt decided to stop treatment due to travel constraints, but at 17 months off treatment had not progressed. Based on this response, 16 pts were enrolled in the CRC cohort of the phase II trial (3 rectal, 13 colon), all heavily pretreated (median: 6 lines of prior therapy, range: 3-14). Median pt age was 64 years (range: 46-80 years); all had an ECOG PS of 1. Median time on study was 2 cycles (range: 1 to 5 cycles) including 5 pts who came off study for early clinical progression. ORR for this cohort was 6% (1 PR). Nine pts had KRAS mutant disease, including the partial responder, who also was the only pt (of the 10 tested by tumor IHC) with MGMT-negative disease. Grade 3 and 4 protocol treatment-related toxicities included thrombocytopenia (2 pts), hemolysis (1 pt), and diarrhea (1 pt). No pts discontinued the protocol because of toxicity and only 1 pt required dose reduction (thrombocytopenia). Conclusions: Despite a promising phase I result from a pt with CRC, a phase II study of combined TRC102 and TMZ treatment in patients with metastatic colon or rectal adenocarcinoma displayed an ORR of 6%. Assessing tumor MGMT status may help identify pts who could benefit from this combination and pharmacodynamic studies are underway to assess drug activity in responding and non-responding patients. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Geraldine OSullivan Coyne, Cecilia Monge Bonilla, Jennifer Zlott, Naoko Takebe, Robert Meehan, Lamin Juwara, Richard Piekarz, Laurence Rubinstein, Deborah F. Wilsker, Robert J. Kinders, Ralph E. Parchment, Shahanawaz Jiwani, Shivaani Kumar, James H. Doroshow, Alice P. Chen. A Phase II trial of TRC102 (methoxyamine HCl) in combination with temozolomide in patients with relapsed metastatic colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-293.

Background: Wee1 tyrosine kinase phosphorylates and inactivates cyclin dependent kinase (Cdk) 1 as part of DNA damage response (DDR) signaling, resulting in G2 cell cycle arrest to enable DNA repair. We are conducting a Phase I study of oral Wee1 inhibitor AZD1775 in patients (pts) with advanced solid tumors using twice-daily (Arm A) and once-daily (Arm B) dosing. Results for Arm A were published previously. Here, we report updated pharmacodynamic (PD), pharmacokinetic (PK), and clinical response results for Arm B. Methods: In Arm B, AZD1775 was administered once daily on days 1-5 and 8-12 of each 21-day cycle in a 3+3 design. Primary objectives were to establish safety, tolerability, PK, and MTD. Secondary objectives included determining PD effects on DDR markers in tumor tissue and circulating tumor cells (CTCs), and tumor response (RECIST 1.1). Post-treatment PD effects were established in Arm A. Given the importance of sustained molecular target control in maximizing targeted therapy efficacy, paired tumor biopsies in Arm B were obtained at baseline and day 8 (pre-dose) to assess durability of Wee1 inhibition and downstream PD effects. Immunofluorescence assays were used to measure target inhibition (pY15-Cdk1/2) and DDR markers (e.g., γH2AX). Results: Arm B included 42 pts; of the 34 evaluable for response, 6 (18%) had a partial response (PR; 4 ovarian, 2 endometrial), and 20 (59%) had stable disease (SD; 2-26 cycles, mean 7). Of 10 evaluable BRCA-deficient pts, 2 had a PR (20%) and 6 had SD (60%; mean: 7.17 cycles). AZD1775 was well-tolerated, as previously reported. Three of 11 paired biopsies had elevated baseline pY15-Cdk (8-23% nuclear area positive [NAP]) and showed persistence of target suppression following the dosing break during days 6-8. Of the 8 pts with low baseline pY15-Cdk (0-3% NAP), 5 had elevated pY15-Cdk levels at day 8 (range: 192-1094%), suggesting a target rebound effect. Of the 8 pts with elevated pY15-CdK at baseline or day 8, 4 (50%) achieved a PR. Day 8 γH2AX activation was detected for 3 pts, all who progressed within 2 cycles. Preliminary PK data revealed low plasma drug concentrations at day 8 pre-dose (55 ± 50 nM; n = 4) relative to peak levels at day 5 (1697 ± 1062 nM; n = 9). Conclusions: Daily AZD1775 exhibits antitumor activity in pts with both BRCA-proficient and -deficient ovarian and endometrial cancer. Responses amongst pts with elevated pY15-Cdk levels at baseline or day 8 provide additional clinical/PD evidence for the presumed mechanism of action of AZ1775 on the cell cycle and suggest that levels of pY15-Cdk may be an important correlate of response. In contrast, γH2AX activation on day 8 demonstrates the presence of drug-induced DNA double strand breaks that are not associated with clinical drug activity. PK data confirmed substantially reduced plasma AZD1775 concentrations at day 8. Ongoing genomic and CTC analyses may uncover additional molecular determinants of response. NCT01748825. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Arjun Mittra, Geraldine O'Sullivan Coyne, Shivaani Kummar, Khanh Do, Ashley Bruns, Lamin Juwara, Richard Piekarz, Larry Rubinstein, Sheila Prindiville, Elad Sharon, Howard Streicher, Tiffaney Hsia, Ganesh Mugundu, Jiuping Ji, Deborah Wilsker, Angie B. Dull, Brandon Miller, Robert J. Kinders, Ralph Parchment, James H. Doroshow, Alice P. Chen, Naoko Takebe. DNA damage response and therapeutic activity following once-daily administration of the Wee 1 inhibitor AZD1775 (adavosertib) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT099.

2629 Background: Immune checkpoint inhibitors promote antitumor immune responses by enhancing T-cell activity. Measuring the pharmacodynamic effects of these drugs is challenging, as it requires assessing both immune cell and cancer cell populations. To evaluate T cell activation in tumor tissue from patient biopsies, we developed a robust multiplexed immunofluorescence assay. Methods: Our assay uses novel oligo-conjugated antibodies (Ultivue) for simultaneous quantitation of TCR activation (phospho-CD3zeta), immune checkpoint signaling via PD-1 (p-SHP1/p-SHP2), and the net stimulation/inhibition resulting from the integration of these two pathways in CD8 cells (p-ZAP70), while also providing the proximity of CD8 cells to tumor tissues, identified by β-catenin. The method was clinically validated using custom tissue microarrays (TMA) containing tumor biopsies of 3 different histologies (CRC, NSCLC, and breast). Results: From a total of 192 tumor core biopsies, 20/64 NSCLC, 9/64 CRC, and 3/65 breast TMA cores were found to have a significant number of CD8+ tumor infiltrating lymphocytes (TILs) at baseline ( > 50 cells in the examined section). In 18 of the 20 NSCLC cores, ≥50% of CD8 cells both inside and outside of the tumor were activated (CD3z-pY142+). In 6/9 CRC cores, ≥50% of CD8+ cells inside tumor tissues were activated, and in 4/9 CRC cores, ≥50% of CD8+ cells in stroma were activated. In 2/3 breast tumor cores, 90% of CD8+ cells inside tumor tissues were activated; in the remaining core, 90% of CD8+ cells in stroma were activated. Interestingly, all 192 cores had minimal to no expression of activated Zap70 (pY493) in CD8+ cells. Conclusions: Depending on tumor histology, baseline biopsy samples may contain variable numbers of activated CD8+ TILs (CD3z-pY142+), which may reside inside or outside of tumor regions and express very low levels of Zap70-pY493. Anti-PD-1 therapy is predicted to enhance T-cell cytotoxic activity, as demonstrated by an increased number of TILs and elevated Zap70-pY493 expression. This assay is being used for pharmacodynamic evaluations in ongoing immunotherapy clinical trials. Funded by NCI Contract No HHSN261200800001E.

3039 Background: Liquid biopsies are emerging as a powerful complement to tumor biopsies for the clinical management of cancer patients. A large gene panel with robust analytical performance that accurately assesses variants, tumor mutational burden (TMB), and microsatellite instability in plasma would be of high value for immunotherapy studies, monitoring minimal residual disease and early cancer detection. To this end, we have completed the initial validation for the cell-free DNA (cfDNA) assay, TruSight Oncology 500 (TSO500), which interrogates the full coding region of 523 genes plus selected intronic regions for fusion detection in 23 driver genes. Methods: Cell-free DNA was extracted from plasma collected from Streck or EDTA blood tubes and quantitated to achieve an assay input of ≥10 ng. Libraries were constructed using unique molecular identifiers (UMIs) and duplex barcodes for error correction, then enriched by target capture and sequenced on a NovaSeq 6000. Healthy donor (HD) specificity assessment used matched white blood cell results to filter germline and clonal hematopoiesis variants. Contrived specimens were used to evaluate sensitivity. Single nucleotide variants (SNVs) (n = 36), insertion/deletions (indels) (n = 19), copy number variants (CNVs) (n = 6), and fusions (n = 5) were tested in 2 multi-site replicates. Results: Sensitivity of detection at 0.5% variant allele fraction (VAF) was > 95% and > 97% for SNVs and indels, respectively. All expected CNVs were identified at the targeted threshold of ≥1.3X change and showed strong correlation with matched digital PCR results. All fusions were identified at ≥0.4% VAF. Specificity in HD was > 99.99%. In 22 temporally matched tumor and blood samples from late-stage patients, 58% of all reportable mutations in tumor were identified in cfDNA. Preliminary TMB analysis identified one TMB high case with POLE p.P286R observed in both tissue and cfDNA. Conclusions: In this initial validation study the TSO500 cfDNA assay exhibited high sensitivity and specificity consistent with requirements for clinical applications. Ongoing studies will further evaluate TSO500 as a complement or potential alternative to tissue biopsy for the genomic profiling of cancer patients.

Summary Background Avelumab (MSB0010718C) is a human IgG1 monoclonal antibody that binds to PD-L1, inhibiting its binding to PD-1, which inactivates T cells. We aimed to establish the safety and pharmacokinetics of avelumab in patients with solid tumours while assessing biological correlatives for future development. Methods This open-label, single-centre, phase 1a, dose-escalation trial (part of the JAVELIN Solid Tumor trial) assessed four doses of avelumab (1 mg/kg, 3 mg/kg, 10 mg/kg, and 20 mg/kg), with dose-level cohort expansions to provide additional safety, pharmacokinetics, and target occupancy data. This study used a standard 3 + 3 cohort design and assigned patients sequentially at trial entry according to the 3 + 3 dose-escalation algorithm and depending on the number of dose-limiting toxicities during the first 3-week assessment period (the primary endpoint). Patient eligibility criteria included age 18 years or older, Eastern Cooperative Oncology Group performance status 0–1, metastatic or locally advanced previously treated solid tumours, and adequate end-organ function. Avelumab was given as a 1-h intravenous infusion every 2 weeks. Patients in the dose-limiting toxicity analysis set were assessed for the primary endpoint of dose-limiting toxicity, and all patients enrolled in the dose-escalation part were assessed for the secondary endpoints of safety (treatment-emergent and treatment-related adverse events according to National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0), pharmacokinetic and pharmacodynamic profiles (immunological effects), best overall response by Response Evaluation Criteria, and antidrug antibody formation. The population for the pharmacokinetic analysis included a subset of patients with rich pharmacokinetic samples from two selected disease-specific expansion cohorts at the same study site who had serum samples obtained at multiple early timepoints. This trial is registered with ClinicalTrials.gov, number NCT01772004. Patient recruitment to the dose-escalation part reported here is closed. Findings Between Jan 31, 2013, and Oct 8, 2014, 53 patients were enrolled (four patients at 1 mg/kg, 13 at 3 mg/kg, 15 at 10 mg/kg, and 21 at 20 mg/kg). 18 patients were analysed in the dose-limiting toxicity analysis set: three at dose level 1 (1 mg/kg), three at dose level 2 (3 mg/kg), six at dose level 3 (10 mg/kg), and six at dose level 4 (20 mg/kg). Only one dose-limiting toxicity occurred, at the 20 mg/kg dose, and thus the maximum tolerated dose was not reached. In all 53 enrolled patients (the safety analysis set), common treatment-related adverse events (occurring in >10% of patients) included fatigue (21 patients [40%]), influenza-like symptoms (11 [21%]), fever (8 [15%]), and chills (6 [11%]). Grade 3–4 treatment-related adverse events occurred in nine (17%) of 53 patients, with autoimmune disorder (n=3), increased blood creatine phosphokinase (n=2), and increased aspartate aminotransferase (n=2) each occurring in more than one patient (autoimmune disorder in two patients at 10 mg/kg and one patient at 20 mg/kg, increased blood creatine phosphokinase in two patients at 20 mg/kg, and increased aspartate aminotransferase in one patient at 1 mg/kg, and one patient at 10 mg/kg). Six (11%) of 53 patients had a serious treatment-related adverse event: autoimmune disorder (two [13%]), lower abdominal pain (one [7%]), fatigue (one [7%]), and influenza-like illness (one [7%]) in three patients treated at 10 mg/kg dose level, and autoimmune disorder (one [5%]), increased amylase (one [5%]), myositis (one [5%]), and dysphonia (one [5%]) in three patients who received the 20 mg/kg dose. We recorded some evidence of clinical activity in various solid tumours, with partial confirmed or unconfirmed responses in four (8%) of 53 patients; 30 (57%) additional patients had stable disease. Pharmacokinetic analysis (n=86) showed a dose-proportional exposure between doses of 3 mg/kg and 20 mg/kg and a half-life of 95–99 h (3·9–4·1 days) at the 10 mg/kg and 20 mg/kg doses. Target occupancy was greater than 90% at doses of 3 mg/kg and 10 mg/kg. Antidrug antibodies were detected in two (4%) of 53 patients. No substantial differences were found in absolute lymphocyte count or multiple immune cell subsets, including those expressing PD-L1, after treatment with avelumab. 31 (58%) of 53 patients in the overall safety population died; no deaths were related to treatment on study. Interpretation Avelumab has an acceptable toxicity profile up to 20 mg/kg and the maximum tolerated dose was not reached. Based on pharmacokinetics, target occupancy, and immunological analysis, we chose 10 mg/kg every 2 weeks as the dose for further development and phase 3 trials are ongoing. Funding National Cancer Institute and Merck KGaA.