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Purpose Patients with recurrent glioblastoma (rGBM) have a poor prognosis. Epidermal growth factor receptor (EGFR) gene amplification is present in ~ 50% of glioblastomas (GBMs). Depatuxizumab mafodotin (depatux-m), formerly ABT-414, is an antibody–drug conjugate that preferentially binds cells with EGFR amplification, is internalized and releases a potent antimicrotubule agent, monomethyl auristatin F (MMAF). Here we report the safety, pharmacokinetics, and efficacy of depatux-m monotherapy at the recommended Phase 2 dose (RPTD) in patients with EGFR-amplified, rGBM. Methods M12-356 (NCT01800695) is an open-label study with three escalation and expansion cohorts. Sixty-six patients with EGFR-amplified, rGBM were treated with depatux-m monotherapy at 1.25 mg/kg intravenously every 2 weeks. Adults with measurable rGBM, who were bevacizumab-naïve, with EGFR amplification were eligible. Results Among 66 patients, median age was 58 years (range 35–80). All patients were previously treated with radiotherapy/temozolomide. The most common adverse events (AEs) were eye related (91%), including blurred vision (65%), dry eye (29%), keratitis, and photophobia (27% each). Grade 3/4 AEs occurred in 42% of all patients, and ocular Grade 3/4 AEs occurred in 33% of patients overall. One patient (2%) had a Grade 4 ocular AE. Ocular AEs were manageable and usually resolved once treatment with depatux-m ceased. The objective response rate was 6.8%, the 6-month progression-free survival rate was 28.8%, and the 6-month overall survival rate was 72.5%. Conclusion Depatux-m monotherapy displayed frequent but mostly Grade 1/2 ocular toxicities. A PFS6 of 28.8% was observed in this rGBM population, warranting further study. Electronic supplementary material The online version of this article (doi:10.1007/s00280-017-3451-1) contains supplementary material, which is available to authorized users.

Objective To review the currently available research into romantic partner involvement during oncology consultations. Methods Studies were identified via database searches plus hand-searching. A narrative review was performed using the principles of Thematic, and Framework syntheses. The search strategy was performed according to the principles of PRISMA. Results From 631 results, 18 studies were included. The findings indicate that romantic partners are most valued by patients when they provide emotional, practical, and informational support. It is also indicated that psychosocial and sexual concerns are rarely discussed. Couples’ self-reported satisfaction with consultations appear related to the extent of romantic partner involvement, the roles that they enacted, and the extent to which psychosocial and sexual concerns were addressed. Conclusion This review indicates that romantic partner involvement during clinical consultations enhances the couple’s experience. However, there are methodological limitations to this body of research, which are discussed in this review. Practice implications Research to date has yet to offer an exploration of the social practices and conversational actions relating to romantic partner involvement during triadic oncology consultations. Future studies that draw upon recordings of these consultations, using methods capable of analysing situated social practices can address this gap.

Background Patients with glioblastoma (GBM) have a dismal prognosis. Nearly all will relapse with no clear standard of care for recurrent disease (rGBM). Approximately 50% of patients have tumors harboring epidermal growth factor receptor (EGFR) amplification. The antibody–drug conjugate depatuxizumab mafodotin (depatux-m) binds cells with EGFR amplification, is internalized, and releases a microtubule toxin, killing the cell. Here we report efficacy, safety and pharmacokinetics (PK) of depatux-m + temozolomide (TMZ) in patients with EGFR-amplified rGBM. Methods M12-356 (NCT01800695) was an open-label study encompassing patients with newly diagnosed or rGBM across 3 treatment arms. Results are reported for adults with EGFR-amplified, measurable rGBM who received depatux-m (0.5–1.5 mg/kg) on days 1 and 15, and TMZ (150–200 mg/m2) on days 1–5 in a 28-day cycle. Patients were bevacizumab and nitrosourea naïve. Results There were 60 patients, median age 56 years (range, 20–79). Fifty-nine patients previously received TMZ. Common adverse events (AEs) were blurred vision (63%), fatigue (38%), and photophobia (35%). Grades 3/4 AEs were split between ocular and non-ocular AEs, occurring in 22% of patients each. Systemic PK exposure of depatux-m was dose proportional. The objective response rate was 14.3%, the 6-month progression-free survival rate was 25.2%, and the 6-month overall survival rate was 69.1%. Conclusions Depatux-m + TMZ displayed an AE profile similar to what was described previously. Antitumor activity in this TMZ-refractory population was encouraging. Continued study of depatux-m in patients with EGFR-amplified, newly diagnosed, or recurrent GBM is ongoing in 2 global, randomized trials (NCT02573324, NCT02343406).

Losatuxizumab vedotin (formerly ABBV-221) is a second-generation antibody-drug conjugate targeting epidermal growth factor receptor (EGFR). In this multicenter phase 1 study, eligible patients with EGFR-dependent solid tumors received losatuxizumab vedotin (3 + 3 design) intravenously at starting dose of 0.3 mg/kg over 3 h per 21-day cycle, with alternate dosing schedules utilized (2 weeks on/1 week off or weekly) to mitigate infusion reactions. Forty-five patients received ≥1 doses of losatuxizumab vedotin (13 colon, 6 non-small cell lung cancer, 5 head and neck [HNC], 5 glioblastoma multiforme, 2 breast, 14 other). Tumor samples were evaluated for EGFR protein expression by immunohistochemistry, EGFR and EGFR ligand mRNA expression by RNAseq, and results compared with outcome. Most common adverse events were infusion-related reaction (22/45; 49%) and fatigue (20/45; 44%). While most infusion reactions were grade ≤ 2, four patients experienced grade ≥3 infusion reactions. Several infusion reaction mitigation strategies were explored. Because of the high incidence of infusion reactions, the trial was stopped and the maximum tolerated dose was not reached. The last cleared dose: 6 mg/kg/cycle. Nineteen patients (42%) had stable disease; 4 remained on study >6 months. One HNC patient with increased levels of EGFR and EGFR ligands (amphiregulin, epiregulin) achieved a confirmed partial response. Pharmacokinetic analysis of losatuxizumab vedotin showed exposures appeared to be approximately dose-proportional. The high frequency of infusion reactions necessitated early closure of this trial. The detailed mitigation strategies used in this protocol for infusion-related reactions may provide beneficial information for trial design of agents with high infusion reaction rates.

Background Epidermal growth factor receptor (EGFR) alterations are associated with multiple cancers. Current EGFR-directed therapies have led to increased efficacy but are associated with specific side effects. The antibody-drug conjugate depatuxizumab mafodotin (depatux-m) targets EGFR with a monoclonal antibody linked to a cytotoxin, and is highly tumor-specific. Methods This phase 1/2 study evaluated the safety, pharmacokinetics, and efficacy of depatux-m in patients who had advanced solid tumors with known wild-type EGFR overexpression, amplification, or mutated EGFR variant III. A 3 + 3 dose escalation was used, and 2 dosing schedules were evaluated. Depatux-m also was manufactured under an alternate process to reduce the drug load and improve the safety profile, and it was tested at the maximum tolerated dose (MTD). In another cohort, prolonged infusion time of depatux-m was evaluated; and a cohort with confirmed EGFR amplification also was evaluated at the MTD. Results Fifty-six patients were treated. The MTD and the recommended phase 2 dose for depatux-m was 3.0 mg/kg. Common adverse events (AEs) were blurred vision (48%) and fatigue (41%). A majority of patients (66%) experienced 1 or more ocular AEs. Grade 3 or 4 AEs were observed in 43% of patients. One patient with EGFR-amplified, triple-negative breast cancer had a partial response. Stable disease was observed in 23% of patients. Pharmacokinetics revealed that depatux-m exposures were approximately dose-proportional. Conclusions Depatux-m resulted in infrequent nonocular AEs but increased ocular AEs. Patient follow-up confirmed that ocular AEs were reversible. Lowering the drug-antibody ratio did not decrease the number of ocular AEs. A partial response in 1 patient with EGFR-amplified disease provides the opportunity to study depatux-m in diseases with a high incidence of EGFR amplification. Cancer 2018;124:2174-83. © 2018 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Epidermal growth factor receptor (EGFR) amplification has been reported to occur in ~ 50% of glioblastomas (GBMs). We are conducting several global studies that require central testing for EGFR amplification during screening, representing an opportunity to confirm the frequency of amplification in GBM in a large cohort and to evaluate whether EGFR amplification differs by region of the world. EGFR amplification was measured by fluorescence in situ hybridization during screening for therapeutic trials of an EGFR antibody–drug conjugate: two Phase 2/3 global trials (INTELLANCE-1, INTELLANCE-2), and a Japanese Phase 1/2 trial (INTELLANCE-J). We evaluated the proportion of tumor tissue samples harboring EGFR amplification among those tested and differences in amplification frequency by geography. EGFR was amplified in 54% of 3150 informative cases screened for INTELLANCE-1 and -2, consistent with historic controls, but was significantly lower in patients from Asia versus the rest of the world (35% vs. 56%, P

Purpose: Patients with glioblastoma (GBM) have a poor prognosis and are in desperate need of better therapies. As therapeutic decisions are increasingly guided by biomarkers, and EGFR abnormalities are common in GBM, thus representing a potential therapeutic target, we systematically evaluated methods of assessing EGFR amplification by multiple assays. Specifically, we evaluated correlation among fluorescence in situ hybridization (FISH), a standard assay for detecting EGFR amplification, with other methods. Experimental Design: Formalin-fixed, paraffin-embedded tumor samples were used for all assays. EGFR amplification was detected using FISH (N = 206) and whole-exome sequencing (WES, N = 74). EGFR mRNA expression was measured using reverse transcription-polymerase chain reaction (RT-PCR, N = 206) and transcriptome profiling (RNAseq, N = 64). EGFR protein expression was determined by immunohistochemistry (IHC, N = 34). Significant correlations among various methods were determined using Cohen's kappa (κ = 0.61–0.80 defines substantial agreement) or R2 statistics. Results: EGFR mRNA expression levels by RNA sequencing (RNAseq) and RT-PCR were highly correlated with EGFR amplification assessed by FISH (κ = 0.702). High concordance was also observed when comparing FISH to WES (κ = 0.739). RNA expression was superior to protein expression in delineating EGFR amplification. Conclusions: Methods for assessing EGFR mRNA expression (RT-PCR, RNAseq) and copy number (WES), but not protein expression (IHC), can be used as surrogates for EGFR amplification (FISH) in GBM. Collectively, our results provide enhanced understanding of available screening options for patients, which may help guide EGFR-targeted therapeutic approaches.

BACKGROUND: Depatuxizumab mafodotin (depatux-m, formerly ABT-414) is an EGFR-directed antibody-drug conjugate being developed for treatment of EGFR-amplified glioblastoma (GBM). As therapeutic pressure engenders tumor adaptations, it is important to understand the stability of biomarkers targeted by precision medicine approaches such as depatux-m. Therefore, we assessed EGFR amplification (amp) and expression in longitudinally-sampled GBMs from patients (pts) treated +/- depatux-m to explore biomarker stability. MATERIAL AND METHODS: Formalin-fixed, paraffin embedded GBM tumor tissue was analyzed from 68 patients who underwent at least 2 surgeries; EGFR amp was detected by in situ hybridization (e.g., FISH) or next-generation sequencing in all samples. Fifty-six pts did not receive depatux-m; among 12 pts who did, EGFR expression was also evaluated by RNA sequencing. RESULTS: Of 56 pts who did not receive depatux-m, 31 (55%) had tumors harboring EGFR amp at 1st surgery (initial diagnosis); among those, EGFR amp was maintained at re-operation in 27 (87%), and not maintained in 4 (13%). None of the 25 cases without baseline EGFR amp acquired it at the 2nd surgery. Of 12 pts treated with depatux-m between surgeries, 9 cases harbored EGFR amp at baseline which was maintained in 4 (44%) at the 2(nd) surgery, all 4 of which had the highest levels of EGFR expression at baseline. Of the 3 cases without EGFR amp at baseline, none acquired amplification at the 2(nd) surgery, and all 3 had the lowest levels of EGFR expression at study start. CONCLUSION: The presence of EGFR amp in GBM tissue at baseline was maintained at 2(nd) surgery in 87% of pts who received treatment other than depatux-m, and in 44% following depatux-m exposure. Therefore, depatux-m exposure appears to reduce EGFR amp maintenance (p=0.0159 by Fisher’s exact test); accordingly, the therapeutic approach may influence EGFR status. In no case was EGFR amp acquired at recurrence, regardless of depatux-m therapy. Ongoing analyses of additional tumor samples will increase power and further examine concordance among EGFR amp assays.

BACKGROUND: We recently reported an acceptable safety and pharmacokinetic profile of depatuxizumab mafodotin (depatux-m), formerly called ABT-414, plus radiation and temozolomide in newly diagnosed glioblastoma (arm A). The purpose of this study was to evaluate the safety and pharmacokinetics of depatux-m, either in combination with temozolomide in newly diagnosed or recurrent glioblastoma (arm B) or as monotherapy in recurrent glioblastoma (arm C). METHODS: In this multicenter phase I dose escalation study, patients received depatux-m (0.5–1.5 mg/kg in arm B, 1.25 mg/kg in arm C) every 2 weeks by intravenous infusion. Maximum tolerated dose (MTD), recommended phase II dose (RP2D), and preliminary efficacy were also determined. RESULTS: Thirty-eight patients were enrolled as of March 1, 2016. The most frequent toxicities were ocular, occurring in 35/38 (92%) patients. Keratitis was the most common grade 3 adverse event observed in 6/38 (16%) patients; thrombocytopenia was the most common grade 4 event seen in 5/38 (13%) patients. The MTD was set at 1.5 mg/kg in arm B and was not reached in arm C. RP2D was declared as 1.25 mg/kg for both arms. Depatux-m demonstrated a linear pharmacokinetic profile. In recurrent glioblastoma patients, the progression-free survival (PFS) rate at 6 months was 30.8% and the median overall survival was 10.7 months. Best Response Assessment in Neuro-Oncology responses were 1 complete and 2 partial responses. CONCLUSION: Depatux-m alone or in combination with temozolomide demonstrated an acceptable safety and pharmacokinetic profile in glioblastoma. Further studies are currently under way to evaluate its efficacy in newly diagnosed (NCT02573324) and recurrent glioblastoma (NCT02343406).

Background Ilorasertib (ABT-348) is a novel inhibitor of Aurora kinase, vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptors, and the Src families of tyrosine kinases. Ilorasertib alone or in combination with azacitidine demonstrated activity in preclinical models in various hematological malignancies, indicating that pan-Aurora kinase and multiple kinase inhibition may have preferential antileukemic activity. This phase 1 trial determined the safety, pharmacokinetics, and preliminary antitumor activity of ilorasertib alone or combined with azacitidine in advanced hematologic malignancies. Patients and methods Fifty-two patients (median age, 67 years; 35 % with >4 prior regimens) with acute myelogenous leukaemia (AML; n = 38), myelodysplastic syndrome (n = 12), or chronic myelomonocytic leukaemia (n = 2) received 3 or 6 doses of ilorasertib per 28-day cycle and were assigned to arm A (once-weekly oral), B (twice-weekly oral), C (once-weekly oral plus azacitidine), or D (once-weekly intravenous) treatment. Results Maximum tolerated doses were not determined; the recommended phase 2 oral monotherapy doses were 540 mg once weekly and 480 mg twice weekly. The most common grade 3/4 adverse events were hypertension (28.8 %), hypokalemia (15.4 %), anemia (13.5 %), and hypophosphatemia (11.5 %). Oral ilorasertib pharmacokinetics appeared dose proportional, with a 15-hour half-life and no interaction with azacitidine. Ilorasertib inhibited biomarkers for Aurora kinase and VEGF receptors, and demonstrated clinical responses in 3 AML patients. Conclusions Ilorasertib exhibited acceptable safety and pharmacokinetics at or below the recommended phase 2 dose, displayed evidence of dual Aurora kinase and VEGF receptor kinase inhibition, and activity in AML.

Background Venetoclax (ABT-199), a first-in-class orally bioavailable BCL-2-selective inhibitor, was recently approved by the FDA for use in patients with 17p-deleted chronic lymphocytic leukemia who have received prior therapy. It is also being evaluated in numerous clinical trials for treating patients with various hematologic malignancies. As with any targeted cancer therapy, it is critically important to identify potential mechanisms of resistance, both for patient stratification and developing strategies to overcome resistance, either before it develops or as it emerges. Methods In order to gain a more comprehensive insight into the nature of venetoclax resistance mechanisms, we evaluated the changes in the BCL-2 family members at the genetic and expression levels in seven different venetoclax-resistant derived leukemia and lymphoma cell lines. Results Gene and protein expression analyses identified a number of different alterations in the expression of pro- and anti-apoptotic BCL-2 family members. In the resistant derived cells, an increase in either or both the anti-apoptotic proteins BCL-XL or MCL-1, which are not targeted by venetoclax was observed, and either concomitant or exclusive with a decrease in one or more pro-apoptotic proteins. In addition, mutational analysis also revealed a mutation in the BH3 binding groove (F104L) that could potentially interfere with venetoclax-binding. Not all changes may be causally related to venetoclax resistance and may only be an epiphenomenon. For resistant cell lines showing elevations in BCL-XL or MCL-1, strong synergistic cell killing was observed when venetoclax was combined with either BCL-XL- or MCL-1-selective inhibitors, respectively. This highlights the importance of BCL-XL- and MCL-1 as causally contributing to venetoclax resistance. Conclusions Overall our study identified numerous changes in multiple resistant lines; the changes were neither mutually exclusive nor universal across the cell lines tested, thus exemplifying the complexity and heterogeneity of potential resistance mechanisms. Identifying and evaluating their contribution has important implications for both patient selection and the rational development of strategies to overcome resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3383-5) contains supplementary material, which is available to authorized users.

Background. The purpose of this study was to determine the maximum tolerated dose (MTD), recommended phase II dose (RPTD), safety, and pharmacokinetics of ABT-414 plus radiation and temozolomide in newly diagnosed glioblastoma. ABT-414 is a first-in-class, tumor-specific antibody-drug conjugate that preferentially targets tumors expressing overactive epidermal growth factor receptor (EGFR). Methods. In this multicenter phase I study, patients received 0.5-3.2 mg/kg ABT-414 every 2 weeks by intravenous infusion. EGFR alterations, O 6 -methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation, and isocitrate dehydrogenase (IDH1) gene mutations were assessed in patient tumors. Distinct prognostic classes were assigned to patients based on a Molecular Classification Predictor model. Results. As of January 7, 2016, forty-five patients were enrolled to receive ABT-414 plus radiation and temozolomide. The most common treatment emergent adverse events were ocular: blurred vision, dry eye, keratitis, photophobia, and eye pain. Ocular toxicity at any grade occurred in 40 patients and at grades 3/4 in 12 patients. RPTD and MTD were set at 2 mg/kg and 2.4 mg/kg, respectively. Among 38 patients with pretreatment tumor tested centrally, 39% harbored EGFR amplification, of which 73% had EGFRvIII mutation. Among patients with available tumor tissue (n = 30), 30% showed MGMT promoter methylation and none had IDH1 mutations. ABT-414 demonstrated an approximately dose proportional pharmacokinetic profile. The median duration of progression-free survival was 6.1 months; median overall survival has not been reached. Conclusion. ABT-414 plus chemoradiation demonstrated an acceptable safety and pharmacokinetic profile in newly diagnosed glioblastoma. Randomized studies are ongoing to determine efficacy in newly diagnosed (NCT02573324) and recurrent glioblastoma (NCT02343406).

Background: Depatux-M (ABT-414) is an antibody-drug-conjugate consisting of an antibody (ABT-806) bound to the toxin monomethylauristatin-F. A randomized phase II trial on EGFR-amplified recurrent glioblastomas (GBMs) showed an improvement (p=0.06 in the primary analysis, p=0.024 in follow-up analysis) in overall survival in the Depatux-M+TMZ arm when compared to the control arm (CCNU or TMZ). In this study, we performed targeted next generation sequencing and correlated molecular features with response to treatment in order to better identify patients that benefit from the combination. Material and Methods: DNA and RNA was isolated from samples, collected at initial diagnosis, and selected for regions with highest tumor content. Target selection was done using the Trusight 170 gene panel (Illumina) which interrogates somatic variants and copy number, RNA levels and fusion genes in a set of known cancer genes. Variant calling was done using the Illumina Basespace sequence hub. For this trial, patients were eligible with centrally confirmed EGFR amplification, defined as EGFR/CEP 7 (centromere) ratio ≥ 2 in 15% of cells (FISH). Results: DNA and RNA data were generated from 233 and 234 samples respectively (of the 260 study patients). High-copy gene amplification was detected in EGFR (n=202), MDM2 (n=20), MDM4 (n=22), CDK4 (n=24) and CDK6 (n=5) which correlated with high expression levels. With this assay, 17 tumors did not show EGFR copy number (cn) aberrations (cn < 2.8), a further 14 showed copy number changes consistent with trisomy only (2.8< cn < 4). Most EGFR amplified tumors also had additional genetic changes in the EGFR locus including point mutations (111/202), splice variants (132/202, the most common being EGFRvIII n=96) or fusion genes (13/202). Twenty-one samples did not contain additional genetic changes and expressed only EGFRwt. Response (OS) to treatment was not correlated to EGFR gene expression or amplification levels though, since EGFR amplification was a pre-requisite for inclusion, the majority of cases expressed high levels of EGFR (not shown). Preliminary analysis suggests that subjects with EGFR amplification but without EGFRvIII expression had a trend towards superior clinical benefit from Depatux-M +TMZ (median survival 14.3 v. 8.9 and 8.1 months in the Depatux-M +TMZ, TMZ|CCNU and Depatux M arms respectively; HR 0.56, P=0.047 v. Depatux M monotherapy and HR 0.54, P=0.055 v. TMZ|CCNU ). Conclusion: Depatux-M in combination with TMZ showed a trend towards improved OS in EGFR amplified recurrent glioblastoma. This trend may be greater for subjects with an absence of EGFRvIII expression. Citation Format: Pim J. French, Johan M. Kros, Iris de Heer, Marica Eoli, Juan Manuel Sepulvada, Annemiek Walenkamp, Jean-Sebastian Frenel, Alba Brandes, Paul Clement, Michael Weller, Peter Ansell, Jim Looman, Earle Bain, Lisa Roberts-Rapp, Marie Morfouace, Thierry Gorlia, Vassilis Golfinopoulos, Martin van den Bent. Patients with EGFR amplification but without EGFRvIII expression have improved benefit compared to those with EGFRvIII expression in samples of the INTELLANCE2/EORTC1410 randomized phase II trial [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 4886.

Preclinical Research The quest for a well-validated, non-invasive biomarker to aid in clinical decision making has remained elusive in the cancer space over the last 30 years. Much promise has been attached to circulating tumor cells (CTCs) as prognostic, predictive, and pharmacodynamic biomarkers with the potential to eliminate the need for invasive tumor biopsies and improve on the clinical value of other circulating tumor markers. The CellSearch® system (Veridex, LLC., Raritan, NJ, USA) cleared the U.S. Food and Drug Administration when the presence of CTCs was shown to have prognostic significance in patients with breast, prostate, and colorectal cancer. However, CTCs are not, at present, routinely being used in the clinic to guide treatment decisions. This paper discusses key attributes that a biomarker must possess, the status of other potential cancer biomarkers, and advancements in the capture and characterization of CTCs that will enable actualization of their potential as a reliable and efficient biomarker of disease diagnosis, progression, and response to therapy in the clinic.

Aim To explore the significance of circulating tumor cells (CTCs) detection in the course of preoperative chemotherapy (PC) and their effect on the outcomes. Methods Fifty-five patients with stage II/III invasive breast cancer were enrolled into a preoperative clinical trial. Patients were given PC with sequential single-agent doxorubicin and paclitaxel vs paclitaxel followed by doxorubicin. Blood samples (8 mL) were collected from patients before PC, after each phase, and at 6 months intervals during follow-up. Peripheral blood mononuclear cells were isolated and enriched for epithelial cells. Quantitative RT-PCR was used to determine the presence of cytokeratin 19 (CK19) mRNA. Samples were considered positive when the PCR curve crossed the standard threshold curve. Results After the first phase of chemotherapy, there was a 59% overall reduction in the median tumor volume. The percentage of volume reduction did not differ between patients who presented with detectable CTCs at baseline and those who did not (p=0.89). After the second phase of chemotherapy, there was a further decrease in the median tumor volume to 93% from baseline. There was no correlation between the lack of response and the presence of CTCs either after the first (p=0.36) or second (p=0.5391) phases of PC. The presence of CTCs was a predictor of local or distant relapse (p=0.0411). The detection of CTCs did not affect overall survival (p=0.2569). Conclusion CTCs can be used as predictors of relapse after definitive treatment of locally advanced breast cancer; however, CTCs detection in peripheral blood during the course of PC does not implicate a particular pattern of response to treatment.

An increasing number of BET family protein inhibitors have recently entered clinical trials. It has been reported that attempts of monitoring target engagement of the BET bromodomain inhibitor OTX015 using literature-described putative pharmacodynamic markers, such as c-Myc, BRD2, etc., failed to detect pharmacodynamic marker responses in AML patients treated at active dose and those with clinical responses. Here, we report the identification and characterization of HEXIM1 and other genes as robust pharmacodynamic markers for BET inhibitors. Global gene expression profiling studies were carried out using cancer cells and surrogate tissues, such as whole blood and skin, to identify genes that are modulated by BET family proteins. Candidate markers were further characterized for concentration- and time-dependent responses to the BET inhibitor ABBV-075 in vitro and in vivo. HEXIM1 was found to be the only gene that exhibited robust and consistent modulation by BET inhibitors across multiple cancer indications and surrogate tissues. Markers such as SERPINI1, ZCCHC24, and ZMYND8 were modulated by ABBV-075 and other BET inhibitors across cancer cell lines and xenograft tumors but not in blood and skin. Significant downregulation of c-Myc, a well-publicized target of BET inhibitors, was largely restricted to hematologic cancer cell lines. Incorporating well-characterized pharmacodynamic markers, such as HEXIM1 and other genes described here, can provide a better understanding of potential efficacy and toxicity associated with inhibiting BET family proteins and informs early clinical decisions on BET inhibitor development programs. Mol Cancer Ther; 16(2); 388–96. ©2016 AACR.

BACKGROUND: Depatuxizumab mafodotin (depatux-m) is an antibody-drug conjugate (ADC) composed of an EGFR-targeted antibody conjugated to a microtubule inhibitor warhead, monomethyl auristatin F. In a Phase 1 study in glioblastoma (GBM), selection for EGFR amplification (amp) enriched for patients (pts) likely to respond. We sought to identify other determinants of sensitivity. MATERIAL AND METHODS: We identified an index case with prolonged disease control but unexpectedly without EGFR amp. Whole exome sequencing (WES) of pre-treatment archival tissue identified a point mutation in TUBA3C (tubulin alpha 3c) which mechanistically affects sensitivity to the microtubule-directed warhead of depatux-m, as opposed to the EGFR-targeted antibody. WES analysis, with a focus on the tubulin gene family, was expanded to 48 pts with sufficient archival tissue. In vitro synthetic lethality experiments were performed to confirm the hypothesized importance of tubulin mutations on depatux-m sensitivity. RESULTS: WES revealed some tubulin genes were differentially mutated in depatux-m responders (n = 8) vs non-responders (n = 24), all with EGFR amp. TPPP2, TTLL11, TTLL6, TTLL7, TUBA3C, TUBA3D, TUBAL3, and TUBGCP4 mutations (1–2 per tumor) were observed preferentially in responders; gene knockdown enhanced response in vitro, consistent with mechanistic relevance to warhead sensitivity. TTLL2, TTLL4, TUBB2A, TUBB2B, TUBG1, TUBGCP2, TUBGCP5, TBCC, and TBCE mutations (1–3 per tumor) were observed exclusively in non-responders. CONCLUSION: Response to ADCs depends not only on the presence of the target antigen but also on sensitivity to the conjugated toxin. We identified tubulin gene family mutations correlating with response to the microtubule inhibitor warhead of the ADC depatux-m. While verification of this observation in an independent cohort is required, other experiments to elucidate the mechanism are ongoing. Further data will be presented. A proposed investigator-initiated trial encompassing tubulin mutations as an eligibility criterion is in development. These data related to warhead sensitivity of depatux-m may serve as a guide to support rational drug combination therapy in GBM.

2033Background: Depatuxizumab mafodotin (depatux-m, formerly ABT-414) is an EGFR-directed antibody-drug conjugate being developed for treatment of EGFR-amplified glioblastoma (GBM). As therapeutic ...

Background: Aberrant EGFR plays a vital role in the oncogenesis of multiple tumor types. ABBV-221 is a 2nd-generation antibody-drug conjugate (ADC) targeting EGFR, based on the 1st-generation ADC depatuxizumab mafodotin (depatux-m, formerly ABT-414). Depatux-m demonstrates efficacy in glioblastoma (GBM) patients (pts) with EGFR amplification in ongoing studies. ABBV-221 is an affinity matured monoclonal antibody against EGFR linked to the microtubule toxin monomethylauristatin E. ABBV-221 has higher affinity for EGFR than depatux-m, potentially allowing it to target a broader range of tumor types. To understand the population most suitable for ABBV-221, molecular assays characterizing EGFR pathway status, including EGFR protein, EGFR mRNA, and EGFR ligand mRNA expression, were evaluated from available tumor tissue of enrolled patients. The results of each assay were correlated with patient outcome to determine which marker may be best associated with prognosis. Methods: In this phase 1, multicenter study of ABBV-221, eligible pts have solid tumors that are likely to overexpress EGFR. EGFR protein expression in tumor tissue samples was analyzed with immunohistochemistry (IHC) using the Dako pharmDx IHC assay. EGFR and mRNA expression from the EGFR ligands epiregulin (EREG) and amphiregulin (AREG) were measured using next-generation RNA sequencing (RNAseq). Results: As of 29 March 2017, 42 pts were treated (13 colon, 5 head & neck [H&N] cancer, 5 non-small cell lung cancer, 5 GBM, 2 breast, 12 other). Ten dose escalation cohorts have been completed with the last cleared dose 4.5 mg/kg per cycle. The most common adverse events were infusion reactions in 19/42 pts (45%) and fatigue in 17/42 pts (41%). Sixteen pts (38%) had stable disease (SD), including 4 pts who remained on study longer than 6 months. All markers are expressed in a wide range across enrolled pts. The pts with response had high EGFR and EGFR ligand. One pt with head and neck cancer who received 5 cycles of ABBV-221 had a partial response and elevated levels of both EGFR (3-fold higher than median for all pts) and EGFR ligand (8-fold higher AREG and 19-fold higher EREG than median for all pts). One colorectal cancer pt with durable SD >6 months had high levels of ligand (EREG) but not receptor, and one H&N pt with SD >6 months had high levels of EGFR receptor but not ligand. Conclusions: Multiple EGFR pathway status biomarkers were evaluated and correlated with clinical outcomes observed in this phase 1 trial of ABBV-221. Patients who observed clinical benefit had high levels of EGFR pathway markers. The duration of SD in pts with refractory solid tumors is encouraging. Clinical trial information: NCT02365662 Citation Format: Emiliano Calvo, James M. Cleary, Victor Moreno, Maryella Gifford, Lisa Roberts-Rapp, Peter J. Ansell, Ho-Jin Lee, Beibei Hu, David Barch, Christopher Ocampo, Anthony W. Tolcher. Biomarker analysis in a phase 1 study of the antibody-drug conjugate ABBV-221 in patients with solid tumors likely to overexpress the epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A028.

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2 positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

The anti-apoptotic proteins BCL-2 and MCL-1 have been shown to promote multiple myeloma (MM) cell survival. Venetoclax (VEN) is a potent, selective, and orally bioavailable small-molecule inhibitor of BCL-2. Bortezomib (BTZ) is a proteasome inhibitor that can inhibit MCL-1 activity by increasing the MCL-1 antagonist, NOXA. Results presented herein describe correlative biomarker analyses in the ongoing phase 1b study of VEN in combination with BTZ and dexamethasone in patients with relapsed/refractory (R/R) MM (NCT01794507). As of 19 Aug 2016, 66 patients were enrolled on study. Baseline bone marrow aspirate samples were available from 52 patients, of which 45 were evaluable for BCL-2 family gene expression by droplet digital PCR in CD138-selected tumor cells. Correlation between BCL2 (BCL-2), BCL2L1 (BCL-XL) and MCL1 (MCL-1) mRNA expression (log2-transformed copies/ul normalized to housekeeping gene) and preliminary efficacy [overall response rate (ORR), time to disease progression (TTP) and duration of response (DoR)] were examined by Log-rank and Wilcoxon tests for binary biomarkers, and by risk ratio from Cox proportional hazard model for continuous biomarkers. The ORR was 68% (44/65) for all evaluable patients and 89% (31/35) in patients who had 1-3 prior therapies (31/35). A broad range of BCL2, BCL2L1 and MCL1 expression was observed, however higher BCL2 levels were detected in patients who achieved a partial response (PR) or better (median: 3.01 vs 0.87, p Citation Format: Jeremy Ross, Brenda Chyla, Rasna Goswami, Lisa Roberts-Rapp, Yan Sun, Yanwen Jiang, Elizabeth Punnoose, Maria Verdugo, Anahita Bhathena, Paulo Maciag. BCL2 expression is a potential predictive biomarker of response to venetoclax in combination with bortezomib and dexamethasone in patients with relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT122. doi:10.1158/1538-7445.AM2017-CT122

Selective BCL-2 targeting with venetoclax (VEN) has shown potent anti-tumor activity in human multiple myeloma (MM) cell lines and primary MM cells harboring the t(11;14) translocation, which is present in approximately 15-20% of patients (pts) with MM. Preclinical studies also indicated that VEN single-agent activity correlated with higher expression levels of BCL-2 relative to BCL-XL and MCL-1. The results presented herein describe correlative biomarker analyses in the ongoing phase 1 study of VEN monotherapy in pts with relapsed/refractory (R/R) MM (NCT01794520). As of 19 Aug 2016, 66 pts were enrolled on study, including 30 pts with t(11;14) MM as determined by interphase FISH analysis on CD138-selected bone marrow plasma cells. The overall response rate (ORR) in pts with t(11;14) MM was 40%, compared to 6% in the non-t(11;14) group (n=36). Responses in t(11;14) pts were durable, with a median duration of response (DoR) of 9.7 months and a median time to progression (TTP) of 6.6 months. In contrast, the median TTP was 1.9 months for non-t(11;14) pts. VEN activity was independent of refractory status to prior therapies and clinical responses were observed in t(11;14) pts with concurrent high-risk cytogenetics, including chromosome 17p deletion. In addition to cytogenetic analyses, baseline bone marrow aspirate samples from 44 pts were evaluable for BCL2 (BCL-2), BCL2L1 (BCL-XL) and MCL1 (MCL-1) mRNA expression by droplet digital PCR. Gene expression analysis revealed that the ratios of BCL2:BCL2L1 (log2 median: 2.75 vs 0.56, p low BCL2L1 > high BCL2:MCL1 > high BCL2 > low MCL1. On the basis of these results, bootstrapping and aggregating thresholds from trees was used to estimate a threshold value for BCL2:BCL2L1 that would provide optimum selection of pts likely to have a response. A high BCL2:BCL2L1 expression ratio (≥5.0) was observed in 10/44 (23%) pts, and was enriched in the t(11;14) subgroup [9/24 (38%) vs 1/20 (5%) in the non-t(11;14) subgroup]. Within the t(11;14) group, 8/9 pts with a high BCL2:BCL2L1 expression ratio achieved a partial response or better (ORR 88%), with a median DoR of 9.7 months and median TTP of 11.5 months. Median TTP for t(11;14) pts (n=15) with a low BCL2:BCL2L1 ratio was 5.3 months. Commensurate with preclinical findings, VEN has promising single-agent activity in pts with R/R t(11;14) MM. Additionally, BCL-2 family expression profiling can further define clinical activity within t(11;14) pts, including 88% ORR in high BCL2:BCL2L1 expressors, thus providing further supportive evidence for VEN monotherapy as a unique biomarker-driven approach for MM treatment. Citation Format: Jeremy Ross, Brenda Chyla, Rasna Goswami, Lisa Roberts-Rapp, Yan Sun, Yanwen Jiang, Elizabeth Punnoose, Maria Verdugo, Anahita Bhathena, Paulo Maciag. Correlative biomarkers of response to venetoclax monotherapy in patients with relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT121. doi:10.1158/1538-7445.AM2017-CT121

2003 Background: GBM is the most common malignant primary brain tumor in adults. Pts with rGBM have a poor prognosis. EGFR is amplified (amp) in ~50% of GBMs and is a compelling therapeutic target. ABT-414 is an antibody-drug conjugate composed of an EGFR-directed antibody conjugated to a microtubule toxin, MMAF. ABT-414 binds a unique epitope exposed during EGFR activation, either through ligand stimulation or mutation such as EGFR variant III (EGFRvIII), releasing MMAF into the cancer cell. Here, we report a pooled safety and efficacy analysis of ABT-414 +/- TMZ in EGFR amp, rGBM. Methods: M12-356 is a Phase 1, open-label, multi-arm study. Results from the 2 arms accruing rGBM pts were pooled for analysis. Eligible adults had rGBM, centrally confirmed EGFR amp, and KPS ≥ 70. Pts received 0.5-1.25 mg/kg ABT-414 on days 1 and 15 +/- 150-200 mg/m2 TMZ on days 1-5 of 28-day cycles until progression (per RANO). Results: As of 11 January 2017, 126 pts were treated. The most common adverse events (AEs, ≥ 25% pts) were ocular (90%) and included blurred vision (64%) and photophobia (31%), which were mainly reversible. Common non-ocular AEs were fatigue (36%) and headache (30%). Grade 3/4 AEs (≥ 5% pts) included ocular toxicities (29%) and decreased platelets/thrombocytopenia (10%). Serious AEs included seizure and keratitis (2% each). Of 125 pts evaluable by RANO, 52% had improvement or stabilization as best response (2 CR, 9 PR, 54 SD), and the remaining 60 (48%) had PD. Of 115 pts with measurable disease at baseline, the objective response rate (ORR) was 10% (2 CR + 9 PR). For 5 pts, re-resection for radiographic PD revealed mostly necrotic tissue and pts were classified as SD, suggesting the ORR may be an underestimate. Of all 126 pts, the 6-month PFS rate (PFS6) was 26%; median OS was 8.5 months. Conclusions: In this Phase 1 trial of EGFR amp, rGBM, we observed encouraging disease control (52%, CR + PR + SD) and PFS6 (26%) rates. Toxicity was mainly ocular, and reversible. A global, randomized trial of ABT-414 vs. ABT-414 + TMZ vs. TMZ/lomustine in EGFR amp, rGBM has completed accrual with results expected later this year (NCT02343406). Clinical trial information: NCT01800695.

2510 Background: ABBV-221 is a 2nd-generation antibody-drug conjugate (ADC) targeting EGFR based on the 1st-generation ADC ABT-414. ABT-414 shows efficacy in glioblastoma (GBM) patients (pts) with EGFR amplification in ongoing studies. ABBV-221 is an affinity matured monoclonal antibody against EGFR linked to the toxin MMAE. ABBV-221 has higher affinity for overexpressed EGFR than ABT-414, potentially allowing it to target a broader range of tumor types. Methods: This is a Phase 1, multicenter study to determine maximum tolerated dose (MTD) and recommended Phase 2 dose (RPTD) of ABBV-221. Pts are required to have an EGFR-dependent cancer to be eligible. Starting dose of ABBV-221: 0.3 mg/kg IV infused over 3 hrs for each 21-day cycle, with alternate dosing schedules utilized (2 wks on, 1 wk off or weekly) to mitigate infusion reactions. Results: As of 11 January 2017, 42 pts were treated (13 colon, 5 head & neck (H&N) cancer, 5 non-small cell lung cancer, 5 GBM, 2 breast, 12 other). Ten dose escalation cohorts have been completed with the last cleared dose 4.5 mg/kg per cycle. Tumor tissue samples were evaluated for EGFR protein expression by IHC, EGFR and EGFR ligand mRNA expression by RNAseq, and the results compared to outcome. The most common adverse event (AE) was infusion reaction in 18/42 pts (43%); 3 pts experienced severe infusion reactions. Several mitigation strategies were used to permit continued dose escalation. The other most common AE was fatigue in 17/42 pts (41%). Only 1 pt had keratitis (Grade 4). Sixteen pts (38%) had stable disease (SD), including 4 pts who remained on study longer than 6 months. One H&N pt who has received 2 cycles of ABBV-221 had an unconfirmed partial response and continues to be treated. This pt had high levels of both EGFR and EGFR ligand. Preliminary pharmacokinetics (PK) analysis suggests ABBV-221 exposures are approximately dose-proportional. Conclusions: Safety, PK, pharmacodynamics, and preliminary efficacy data of ABBV-221 warrant further study in this population. Infusion reactions have been manageable and primarily a first dose phenomenon. The duration of SD in pts with refractory solid tumors is encouraging. Clinical trial information: NCT02365662.

Bromodomain and extra-terminal (BET) family are dual bromodomain-containing proteins that play an important role in transcription regulation. ABBV-075 is a small molecule BET bromodomain inhibitor currently in phase 1 clinical trials. Here we report the identification of Hexim1 and other BET-responsive genes as robust pharmacodynamic markers for monitoring ABBV-075 target engagement in tumors and in surrogate tissues such as whole blood and skin. Transcription profiling of cancer cell lines for their responses to BET inhibitors identified a set of 9 BET-regulated genes, including Hexim1, that responded to BET inhibition across cancer cell lines and in xenograft tumors. Further characterization of BET-responsive genes in surrogate tissues such as mouse skin, mouse/human PBMCs and whole blood revealed that the mRNA level of Hexim1 exhibited the best response to BETi treatment across different settings. A dose-dependent increase of Hexim1 expression was detected in the whole blood of ABBV-075 treated tumor bearing mice, and the Hexim1 response was closely correlated with the plasma drug concentration and largely reflected the anti-tumor efficacy at various dose levels of ABBV-075. Hexim1 is part of a protein/RNA complex that sequesters pTEFb and prevents its recruitment by BRD4. It also reportedly mediates the anti-proliferative activity of BRD4 in AML. Taken together, Hexim1 could serve as a functional relevant pharmacodynamic marker for monitoring ABBV-075 target engagement in animal models and in the clinical setting. Citation Format: Xiaoli Huang, Xiaoyu Lin, Leiming Li, Rongqi Wang, Lisa Roberts, Paul Hessler, Tamar Uziel, Lloyd Lam, Terry Magoc, Daniel H. Albert, Steven W. Elmore, Guowei Fang, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3770.

Introduction: Multiple myeloma (MM) tumor cell survival is largely dependent on a complex network of interactions between pro-survival (BCL-2, BCL-XL, MCL-1) and pro-death (BIM, NOXA, BID, BAD, BAK, BAX) BCL-2 family members. Venetoclax (VEN) is a potent, selective, orally bioavailable small-molecule BCL-2 inhibitor. Bortezomib (BTZ) is a proteasome inhibitor that can impede MCL-1 activity by increasing the MCL-1 antagonist NOXA. The addition of VEN has been shown to enhance the activity of BTZ in MM cell line and xenograft models. Chromosomal abnormalities (immunoglobulin heavy chain translocations, amplification, deletions) can also influence sensitivity of MM cells to treatment. Results presented herein describe retrospective biomarker analysis in a subset of dose-escalation patients (pts) from an ongoing phase 1b study of VEN in combination with BTZ and dexamethasone in relapsed/refractory (R/R) MM. Methods: Exploratory objectives included evaluation of chromosomal abnormalities, BCL-2 family member gene expression levels, and ex vivosensitivity of pt MM tumor cells to VEN and/or BTZ. Interphase FISH analysis was performed on CD138-positively selected bone marrow mononuclear cells (BMMC) using probes for t(11;14), t(4;14), deletion 17p (17p-), deletion 13q (13q-), and amplification of chromosomes 5, 9, or 15 (denoted as hyperdiploid [HY]). Absolute quantitation of BCL-2 family members (BCL-2, BCL-XL, MCL-1, BCL-2A1, BIM, BID, BAD, BAX, and NOXA) was performed on CD138-selected BMMCs collected at baseline using TaqMan probe-based droplet digital polymerase chain reaction. Ex vivosensitivity of BMMCs collected at baseline to increasing doses of VEN and/or BTZ was determined by flow cytometry using a combined analysis for the loss of CD138 surface expression and the alteration of cellular morphology (lower FCS). Correlations between biomarkers and median best percentage change in M-protein were examined by Spearman test. Correlations between biomarkers and median time on study (mToS) were examined by log-rank and Wilcoxon tests for binary biomarkers, and by risk ratio from a Cox proportional hazard model for continuous biomarkers. No correction for multiple testing was performed. Results: Forty-one pts were enrolled as of 06/15/2015. The biomarker study subgroups were similar to the overall pt population with respect to demographics. Of the 37/41 (90%) pts assessed by FISH, 5 were t(11;14), 3 were t(4;14), 20 were 13q-, 10 were 17p-, and 15 were HY. In pts with HY, median best percentage change in M-protein was -79.4% compared to +101.65% in non-HY, while mToS was 309 days compared to 29 days in non-HY (p Conclusions: VEN in combination with BTZ and dexamethasone is active in R/R MM. CD138-enriched BMMCs were obtained in sufficient numbers to enable downstream FISH analysis, BCL-2 family gene expression profiling, and ex vivo sensitivity evaluation. The exploratory biomarker analysis showed that higher BCL-2 gene expression levels at baseline significantly correlated with clinical responses to therapy, with a trend toward increased mToS. Additionally, MM cells treated ex vivo demonstrated greater sensitivity to the combination of VEN/BTZ than to the 2 single agents alone. Further investigation of this dataset continues, including analysis of on-treatment tumor samples in a subset of pts. Disclosures Ross: AbbVie: Employment, Equity Ownership. Off Label Use: Venetoclax is an investigational drug that is not yet approved in this indication.. Chyla:AbbVie: Employment, Equity Ownership. Roberts-Rapp:AbbVie: Employment, Equity Ownership. Goswami:AbbVie: Employment, Equity Ownership. Yan:AbbVie: Employment, Equity Ownership. Huang:AbbVie: Employment, Equity Ownership. Bhathena:AbbVie: Employment, Equity Ownership. Enschede:AbbVie: Employment, Equity Ownership. Maciag:AbbVie: Employment, Equity Ownership. Humerickhouse:AbbVie: Employment, Equity Ownership. McKeegan:AbbVie: Employment, Equity Ownership.

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2-positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

Background Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. However, background expression of 'tumor specific markers' in peripheral blood mononuclear cells (PBMC) may confound these studies. The goal of this study was to identify the origin of Cytokeratin 19 (CK19) and HER-2 signal in PBMC and suggest an approach to enhance techniques involved in detection of CTC in breast cancer patients. Methods PBMC from healthy donors were isolated and fractionated into monocytes, lymphocytes, natural killer cells/granulocytes and epithelial populations using immunomagnetic selection and fluorescent cell-sorting for each cell type. RNA isolated from each fraction was analyzed for CK19, HER2 and Beta 2 microglobulin (B2M) using real-time qRT-PCR. Positive selection for epithelial cells and negative selection for NK/granulocytes were used in an attempt to reduce background expression of CK19 and HER2 markers. Results In normal PBMC, CK19 was expressed in the lymphocyte population while HER-2 expression was highest in the NK/granulocyte population. Immunomagnetic selection for epithelial cells reduced background CK19 signal to a frequency of Conclusion We present a novel method to improve the specificity of the traditional method of detecting CTC by identifying the source of the background signals and reducing them by negative immunoselection. Further studies are warranted to improve sensitivity and specificity of methods of detecting CTC will prove to be useful tools for clinicians in determining prognosis and monitoring treatment responses of breast cancer patients.

BACKGROUND: ABT-414 is an EGFR-targeted antibody conjugated to the toxic antimicrotubule agent monomethylauristatin F. In an ongoing phase I study (abstract by Gan et al), all confirmed responses as determined by RANO criteria have been restricted to patients whose tumors harbor EGFR amplification. Genomic analysis was performed to explore mechanisms associated with innate sensitivity and acquired resistance. METHODS: Whole exome sequencing (WES) of diagnostic tumor samples from relapsed glioblastoma patients treated with ABT-414 + temozolomide (TMZ) or ABT-414 alone was performed. WES data were correlated with EGFR amplification and EGFRvIII as determined by FISH and RT-PCR, respectively. To explore acquired resistance, tissue was collected prior to starting ABT-414/TMZ from a patient who had a complete response and again postrelapse. WES and RNAseq of collected tissue were performed to identify mutations and associated transcriptional changes occurring at relapse. RESULTS: WES has been completed for 13 patients (14 samples) and is ongoing for 11 additional samples. Of the 9 samples determined to be EGFR amplified by FISH, 8 were amplified by WES. All 5 samples determined to be EGFRvIII positive by RT-PCR were positive by WES. In the patient with serial tumor collection, EGFR amplification and EGFRvIII expression was not significantly altered postprogression. Mutations in 25 genes were detected only in the postrelapse biopsy including in TOP2A and TOPORS, both of which are reported to be associated with chemotherapeutic resistance. RNAseq demonstrated a 1.7-fold upregulation of TUBGCP4 at the time of relapse. Given the involvement of TUBGCP4 in tubulin biology, this may potentially influence sensitivity to ABT-414's microtubule toxin. CONCLUSIONS: To date, EGFR amplification is necessary but not sufficient for response to ABT-414. Genomic profiling of longitudinally collected tissue may suggest factors involved in treatment sensitivity and acquired resistance. Ongoing mutation analysis in additional samples will be presented.

As a population, non-Hodgkin’s lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2High) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2Low) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

BACKGROUND: Aberrant EGFR signaling plays a vital role in GBM oncogenesis. ABT-414 is an antibody-drug conjugate with a toxic payload (monomethylauristatin F) linked to ABT-806, an antibody targeted to activated EGFR. This phase I, open-label, 3-arm study (NCT01800695) evaluates ABT-414 mono- or combination therapy with TMZ rechallenge in patients with GBM. Herein, we report outcomes in recurrent GBM. METHODS: Eligible patients (≥18 years) had recurrent supratentorial GBM and Karnofsky performance status ≥70. ABT-414 was given intravenously every 2 weeks with TMZ rechallenge (150-200 mg/m2 days 1-5 q28; Arm B) or as monotherapy (Arm C). ABT-414 dose escalation used a modified continual reassessment method. Primary objectives were to assess ABT-414 safety, maximum tolerated dose (MTD), and recommended phase II dose (RPTD); secondary objectives included antitumor activity evaluation. RESULTS: As of April 2015, 19 Arm B patients were treated in 4 dose groups (0.5-1.5 mg/kg), and 27 Arm C patients were treated at 1.25 mg/kg. In Arms B/C, 10/17 patients had amplified EGFR. Most common grade 3/4 treatment-emergent adverse events in either arm were keratitis (11%/19%), γ-glutamyltransferase increase (16%/0%), convulsions (11%/0%), fatigue (11%/0%), and thrombocytopenia (11%/4%). ABT-414 MTD was 1.5 mg/kg in Arm B and was not reached in Arm C; RPTD is 1.25 mg/kg for both arms. Best objective responses were complete response (CR) in 3 patients and a partial response (PR) in 1 patient in Arm B, and 1 CR and 1 PR in Arm C patients (all confirmed responses were observed in patients with EGFR amplification).Objective response rate among EGFR-amplified subjects was 22.2% (6/27). CONCLUSIONS: RPTD for ABT-414 is 1.25 mg/kg for both combination therapy with TMZ rechallenge and monotherapy. ABT-414 demonstrated a unique safety profile including ocular AEs, and appears to have antitumor activity in recurrent GBM with EGFR amplification.

2510 Background: Aberrant EGFR expression and signaling has been identified as a hallmark of cancer growth and survival. ABT-414 is an antibody-drug conjugate composed of the antibody ABT-806, targ...

2016 Background: GBM remains almost universally fatal and new therapies are needed. The epidermal growth factor receptor (EGFR) is a key oncogenic target. ABT-414, an antibody-drug conjugate with a...

ABT-199 is a first-in-class orally bioavailable BCL-2 selective inhibitor that is currently being testing in clinical trials. Clinical activity has been reported in chronic lymphocytic leukemia and NHL patients treated with ABT-199 as a monotherapy. As with any targeted cancer therapy, it is important to identify the potential mechanisms of resistance, not only to inform patient selection but also to develop strategies to circumvent resistance as it emerges. In this study, we generated resistant variants from ABT-199-sensitive cell lines of different leukemia and lymphoma subtypes (two diffuse large B-cell lymphomas, two follicular lymphomas, one leukemia line, and two mantle cell lymphomas) by increasing exposure to ABT-199 in a stepwise manner over time. Compared to the parental cell lines, the variants were 10 to >100-fold less sensitive to ABT-199. The ABT-199-resistant variants were also resistant to ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, as well as to other standard-of-care cancer agents. Gene and protein expression analyses revealed multiple alterations in the expression of BCL-2 family members in the resistant variants; however, not all of the changes were universal. Alterations included changes in the levels of the target protein BCL-2, reduction of the pro-apoptotic proteins BAX, BIM or NOXA, or increases in the anti-apoptotic proteins BCL-XL or MCL-1. In some but not all cases, the changes to gene and protein expression levels were accompanied by changes in DNA copy number. To determine whether changes in the anti-apoptotic BCL-2 family members were causally related to ABT-199 resistance, we tested ABT-199 in combination with BCL-XL and MCL-1 selective inhibitors. Co-treating with ABT-199 and either a BCL-XL or MCL-1 selective compound was synergistic in killing the ABT-199-resistant variants, suggesting these proteins can both contribute directly to ABT-199 resistance. Under appropriate selection pressure, intrinsic traits that mitigate the effects of anticancer drugs can lead to the enrichment of a resistant tumor population. In ABT-199-resistant RS4;11 cells we observed a complete and permanent loss of BAX, one of the ultimate effectors of mitochondrial mediated apoptosis. FACS analysis revealed that there was a pre-existing population of RS4;11 cells lacking this protein. It is likely that these cells had a selective advantage over those expressing BAX when treated with ABT-199. Overall our data indicate that there are distinct mechanisms of resistance to ABT-199, either innate or acquired, which will have important implications for both patient selection and developing strategies to overcome resistance. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for the research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Stephen K. Tahir, Morey L. Smith, Paul Hessler, Lisa Roberts-Rapp, Joel D. Leverson, Lloyd T. Lam. Mechanisms of resistance to ABT-199 in leukemia and lymphoma cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B30.

BACKGROUND: Standard therapies have little effect on the poor survival rates of GBM. Abnormal epidermal growth factor receptor (EGFR) expression and signaling are common in GBM. ABT-414 is a unique antibody-drug conjugate, with a toxic payload (MMAF) targeted to active EGFR or mutant EGFRvIII, that has demonstrated high antitumor activity in preclinical GBM tumor models. METHODS: Objectives were to evaluate the safety, pharmacokinetics (PK), and the maximum tolerated dose (MTD) of ABT-414 when administered with TMZ (recurrent or unresectable GBM, Arm B) or concurrent RT and TMZ (newly diagnosed GBM, Arm A). Adverse events (AEs), PK parameters, objective response (RANO), and tumor tissue EGFR biomarkers were assessed. Dose escalation was determined by the continual reassessment method (CRM). RESULTS: As of April, 2014, 22/21 pts were treated in Arm A/B. Common treatment-emergent AEs included fatigue(n= 11/ 6, Arm A/B); blurred vision(n = 10/9); AST increase(n = 9, Arm A); and nausea(n = 7/7) Grade 3/4 AEs (≥ 2 pts) included lymphopenia(n = 3); eye toxicity(n = 3); brain edema, ALT, AST(n = 2 each) in Arm A and keratitis(n = 2) in Arm B. Doses from 0.5-3.2mg/kg have been explored. Dose limiting toxicities primarily affecting the eye (keratitis) and liver occurred at multiple doses. The CRM has predicted 2.4mg/kg as the MTD in Arm A and 1.25mg/kg in Arm B. Patient samples are being evaluated for EGFR expression, amplification and EGFRvIII status. Efficacy endpoints are not yet mature in Arm A, but 4 patients in Arm B have best responses of 3 PRs and 1 CR. CONCLUSIONS: PK and safety data support doses of 2.4 and 1.25mg/kg as the predicted MTD in Arms A and B. Preliminary safety data demonstrate liver and eye toxicities in Arm A and primarily eye toxicities in Arm B. Updated biomarker results as they correlate with efficacy will be reported. Phase 2 studies assessing ABT-414 in GBM are planned.

Aim: Despite standard therapy for GBM, median survival is 1-2 years. Abnormal epidermal growth factor receptor (EGFR) expression and signaling are common in GBM. ABT-414 is a unique antibody-drug conjugate, with a toxic payload (monomethylauristatin F) targeted to active EGFR or mutant EGFRvIII, that has demonstrated high antitumor activity in preclinical GBM tumor models. Methods: Objectives were to evaluate the safety, pharmacokinetics (PK), and the maximum tolerated dose (MTD) of ABT-414 when administered every 14 days with concurrent RT and TMZ in newly diagnosed GBM. Adverse events, PK parameters, objective response (RANO), and tumor tissue EGFR biomarkers were assessed. Dose escalation was determined by the exposure-adjusted continual reassessment method (EACRM). Results: As of April 9, 2014, 22 pts were treated (13/9 Male/Female, median age 58 years, range 34-79). Common treatment-emergent adverse events (TEAEs, ≥ 5 pts) included fatigue (n= 11); blurred vision (n = 10); thrombocytopenia (n = 8); AST increase (n = 9); ALT increase (n = 8); nausea (n = 7); eye pain (n = 6); lacrimation increase (n = 6); dry eye, headache, and constipation (n = 5 each). Grade 3/4 TEAEs (≥ 2 pts) included lymphopenia (n = 3); eye toxicity (n = 3); brain edema, ALT, AST (n = 2 each). Doses from 0.5 - 3.2 mg/kg have been explored. Dose limiting toxicities primarily affecting the eye (keratitis) and liver occurred at the 2, 2.6, 3.0, and 3.2 mg/kg doses. The EACRM has predicted 2.4 mg/kg as the MTD. Confirmatory safety data are being collected. PK data from 9 subjects in the dose range of 0.5 - 3 mg/kg indicate that exposure (Cmax and AUC) of ABT‐414 appeared to be dose proportional with a half‐life of approximately 11 days. Efficacy endpoints are not yet mature. Patient samples are being evaluated for EGFR expression, amplification and EGFRvIII status to determine which marker best associates with clinical benefit. Conclusions: PK and safety data support a dose of 2.4 mg/kg as the predicted MTD. Preliminary safety data demonstrate increased liver and eye toxicities in addition to common RT + TMZ toxicities. Further follow-up may demonstrate whether ABT-414 improves outcome. Disclosure: H.K. Gan: employee of Ludwig Institute for Cancer Research, which has licensed ABT-806; A. Lassman: Consultancy: Amgen, Celgene, Genentech, Sigma-Tau, Agenus, Roche, Stemline, Synapse, RadMD, Venture Inflections, Novartis, Kyowa Hakko Kirin, Abbott, Scientia Advisors, MSD, GSK, Able Assoc, Defined Health, Easton Assoc.; M.J. van den Bent: Consultancy: Roche, ABBVIE, Actelion, Celldex, AMGEN. Research support: Roche, ABBVIE. Speakers bureau: MSD; A. Scott: employee of Ludwig Institute for Cancer Research, which has licensed ABT-806; Consultancy and stock ownership - Life Science Pharmaceuticals; M. Pedersen, E. Gomez, J. Fischer, W. Ames, H. Xiong, M. Dudley, L. Roberts-Rapp, P.J. Ansell and K. Holen: is an AbbVie employee and may own stock; D.A. Reardon: Speaker's Bureau – Merck/Schering and Genentech/Roche; Advisory Board – Novartis; Amgen; Roche/Genentech; EMD Serono; Stemline Therapeutics; Momenta Pharmaceuticals. All other authors have declared no conflicts of interest.

2021 Background: Patients (pts) with recurrent GBM have few treatment options and a very poor prognosis. GBM tumors often exhibit aberrant epidermal growth factor receptor (EGFR) proliferative sign...