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Adjuvant therapy; Breast cancer; Olaparib Terapia adyuvante; Cáncer de mama; Olaparib Teràpia adjuvant; Càncer de mama; Olaparib Background The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals. Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca.

Representative movie of individual MDA-MB-231LN cells treated with Vehicle, MLN8237, eribulin, or MLN8237/eribulin combination, suspended in collagen, and manually tracked over the course of 23 hours toward the serum chemoattractant-containing reservoir (left side of frame).

File contains Supplementary figures: Figure 1: AUKRA analysis in TNBC cell lines in response to drug treatments. Additional TNBC migration/invasion analysis. Figure 2. Representative FACS analysis flow charts of TNBCs undergoing apoptosis. Figure 3. F-IHC analysis of tumors and lung metastases at the end point of treatment with vehicle, MLN8237, eribulin with indicated phospho-specific antibodies. Figure 4. Representative images of F-IHC, DNA (DAPI) staining of pulmonary tissue (metastases defined by the white outline) and mammary tumors. Figure 5. Fluorescent IHC analysis of metastases using pan-cytokeratin antibodies (Dako, green) to detect human tumor cells in lungs (mouse), DNA-DAPI. Figure 6. WB analysis of LC3BI/II, pAKT, AKT, alpha-tubulin expression in tumor cells isolated mammary tumors and lungs. Figure 7. F-IHC analysis of mammary tumor and lung metastases cells with p62, pAKT antibodies in the presence of Chloroquine. Figure 8. F-IHC staining of pulmonary tissue from, -cleaved Caspase-3, -phosphoAKT1/2. Figure 9. Cell growth analysis of Tumor cells PEN65 derived from PDX treated with indicated drugs. Figure 10. LAESI-MS MS/MS fragmentation of MLN8237 in dosed animal tumor tissue.

File contains detailed information on Supplementary video files.

Table contains diagnostics/characterization of original and patient-derived models.

File contains detailed protocols and information on used reagents to facilitate reproducible of the presented data.

Representative movie of individual BT-549 cells treated with Vehicle, MLN8237, eribulin, or MLN8237/eribulin combination, suspended in collagen, and manually tracked over the course of 23 hours toward the serum chemoattractant-containing reservoir (left side of frame).

Purpose:This open-label, multicenter, phase IB/II study evaluated sapanisertib, a dual inhibitor of mTOR kinase complexes 1/2, plus exemestane or fulvestrant in postmenopausal women with hormone receptor–positive (HR+)/HER2-negative (HER2−) advanced/metastatic breast cancer.Patients and Methods:Eligible patients had previously progressed on everolimus with exemestane/fulvestrant and received ≤3 (phase IB) or ≤1 (phase II) prior chemotherapy regimens. Patients received sapanisertib 3 to 5 mg every day (phase IB), or 4 mg every day (phase II) with exemestane 25 mg every day or fulvestrant 500 mg monthly in 28-day cycles. Phase II enrolled parallel cohorts based on prior response to everolimus. The primary objective of phase II was to evaluate antitumor activity by clinical benefit rate at 16 weeks (CBR-16).Results:Overall, 118 patients enrolled in phase IB (n = 24) and II (n = 94). Five patients in phase IB experienced dose-limiting toxicities, at sapanisertib doses of 5 mg every day (n = 4) and 4 mg every day (n = 1); sapanisertib 4 mg every day was the MTD in combination with exemestane or fulvestrant. In phase II, in everolimus-sensitive versus everolimus-resistant cohorts, CBR-16 was 45% versus 23%, and overall response rate was 8% versus 2%, respectively. The most common adverse events were nausea (52%), fatigue (47%), diarrhea (37%), and hyperglycemia (33%); rash occurred in 17% of patients. Molecular analysis suggested positive association between AKT1 mutation status and best treatment response (complete + partial response; P = 0.0262).Conclusions:Sapanisertib plus exemestane or fulvestrant was well tolerated and exhibited clinical benefit in postmenopausal women with pretreated everolimus-sensitive or everolimus-resistant breast cancer.

Supplementary Material containing Supplementary Methods, 5 Supplementary Figures, and 8 Supplementary Tables

Even though gene amplification or protein overexpression occurs in approximately one-fifth of all breast cancers, the discovery of HER2 has, nevertheless, had profound implications for the disease. Indeed, the characterization of the receptor resulted in a number of significant advances. Structurally, unique features provided avenues for the development of numerous compounds with target-specificity; molecularly, biological constructs revealed a highly complex, internal signal transduction pathway with regulatory effects on tumor proliferation, survival, and perhaps, even resistance; and clinically, disease outcomes manifested its predictive and prognostic value. Yet despite the receptor’s utility, the beneficial effects are diminished by tumor recurrence after neo- or adjuvant therapy as well as losses resulting from the inability to cure patients with metastatic disease. What these observations suggest is that while tumor response may be partially linked to uncoupling cell surface message reception and nuclear gene expression, as well as recruitment of the innate immune system, disease progression and/or resistance may involve a reprogrammable signaling mainframe that elicits alternative growth and survival signals. This review attempts to meld current perceptions related to HER2-positive metastatic breast cancer with particular attention to current biological insights and therapeutic challenges.

Immune checkpoint blockade transformed cancer therapy during the last decade. However, durable responses remain uncommon, early and late relapses occur over the course of treatment, and many patients with PD-L1-expressing tumors do not respond to PD-(L)1 blockade. In addition, while some malignancies exhibit inherent resistance to treatment, others develop adaptations that allow them to evade antitumor immunity after a period of response. It is crucial to understand the pathophysiology of the tumor-immune system interplay and the mechanisms of immune escape in order to circumvent primary and acquired resistance. Here we provide an outline of the most well-defined mechanisms of resistance and shed light on ongoing efforts to reinvigorate immunoreactivity.

PURPOSE Patients with human epidermal growth factor receptor 2 (HER2)–positive metastatic breast cancer eventually develop resistance to dual-antibody therapy with trastuzumab plus pertuzumab. Mechanisms of resistance have not been well elucidated. We evaluated the safety, tolerability, and efficacy of ado-trastuzumab emtansine (T-DM1) plus neratinib in patients who progressed on trastuzumab plus pertuzumab. PATIENTS AND METHODS In this 3 + 3 dose-escalation study, patients with metastatic breast cancer who progressed on trastuzumab, pertuzumab, and a taxane were treated with T-DM1 at 3.6 mg/kg intravenously every 3 weeks and dose-escalating neratinib at 120, 160, 200, or 240 mg/d orally. RESULTS Twenty-seven patients were treated across four dose-levels of neratinib. Dose-limiting toxicity in cycle 1 was grade 3 diarrhea in six patients and grade 3 nausea in one; no patient experienced grade 4 diarrhea, and there were no grade 5 toxicities. Other grade 3 to 4 toxicities included nausea (11%), dehydration (11%), electrolyte abnormality (19%), thrombocytopenia (15%), elevated transaminase levels (7%), and fatigue (7%). Twelve (63%) of 19 evaluable patients had an objective response. Responses occurred at all neratinib doses. Plasma cell–free DNA at baseline showed ERBB2 (HER2) amplification in 10 of 27 patients. Deep and more durable responses occurred in patients with cell-free DNA ERBB2 amplification. Two complete responders had high expression of total HER2 and p95HER2 in baseline tissue. CONCLUSION We report the recommended phase II dose of T-DM1 3.6 mg/kg and neratinib 160 mg/d for this combination. Possible resistance mechanisms to HER2 antibodies may be loss of the HER2 receptor and high expression of p95HER2. These data provide the basis for an ongoing phase II study to better define the activity of this regimen.

Background: TAK-228 is an investigational, oral and highly selective ATP-competitive inhibitor of TORC1/2. Targeting the PI3K/AKT/mTOR pathway with the dual TORC1/2 inhibitor TAK-228 may restore sensitivity to endocrine therapies in patients (pts) with breast cancer who have progressed on the combination of an endocrine agent plus a TORC1 inhibitor. Here we report data from the phase 2 portion of a phase 1b/2 study of TAK-228 plus exemestane (E) or fulvestrant (F). Methods: Postmenopausal women with ER+ and HER2-, inoperable or metastatic breast cancer (MBC) following everolimus (EVE) plus E or F after progression, received oral TAK-228 (4 mg QD) plus E (25 mg QD) or F (500 mg monthly) for 28-day cycles until progressive disease (PD) or unacceptable toxicity (NCT02049957). Pts were enrolled into parallel cohorts based on prior response to EVE plus E or F and were given the same prior therapy (E or F) at their established dose: EVE-sensitive, defined as disease progression after complete response (CR), partial response (PR), or ≥6 mos stable disease (SD); or EVE-resistant, defined as disease progression without a CR or PR, or after Results: From Oct 2015 to Dec 2017, 94 pts were enrolled. Median age was 58 y (range 32–83). At baseline, most pts (67%) had stage IV disease and others were stage IA–IIIC (24%), other (3%) or unknown (5%); 94% of EVE-sensitive (93% E vs 100% F) and 88% of EVE-resistant pts (91% E vs 75% F) had received ≥4 prior lines of therapy. Pts received a median of 3 cycles (1–15) of TAK-228. At data cutoff (24 Apr 2018), 98% of pts had discontinued treatment, mainly due to PD (76%) or adverse events (AEs; 14%). CBR-16 was 41% (n=21) in EVE-sensitive and 26% (n=11) in EVE-resistant pts (table). CBR-24 was 24% in EVE-sensitive (19% E vs 50% F) and 23% in EVE-resistant (23% E vs 25% F) pts. Eleven of 21 pts who achieved CBR-16 also achieved CBR-24 (6 SD, 5 PR) in the EVE-sensitive cohort and 8 of 11 pts in the EVE-resistant cohort (6 SD, 2 PR). The ORR was 12% in EVE-sensitive pts and 9% in EVE-resistant pts (table). Median PFS (95% CI) was 4.1 mos (2.2–5.5) and 3.4 mos (1.9–5.4), and median OS (95% CI) was 15.9 mos (14.1–19.5) and 14.0 mos (13.0–16.0) in the EVE-sensitive and -resistant cohorts, respectively. Drug-related any grade and grade ≥3 AEs were seen in 90% and 29% of pts, respectively. Most common drug-related any grade AEs were nausea (50%), fatigue (38%), hyperglycemia and diarrhea (each 29%); 22% of pts reported a serious AE. No deaths were reported. Treatment is ongoing in two pts. Conclusion: TAK-228 plus E or F showed modest clinical benefit in pts with previously treated, EVE-sensitive or -resistant MBC, with an acceptable safety profile. EVE-sensitive (N=51)EVE-resistant (N=43) TAK-228+TAK-228+Best response, n (%)E (n=43)F (n=8)E (n=35)F (n=8)ORR=CR+PR4 (9)2 (25)3 (9)1 (13)CR001 (3)0PR4 (9)2 (25)2 (6)1 (13)CBR-1617 (40)4 (50)9 (26)2 (25) Citation Format: Diamond JR, Potter D, Salkeni M, Silverman P, Haddad T, Forget F, Awada A, Canon J-L, Danso M, Lortholary A, Bourgeois H, Tan-Chiu E, Patel C, Neuwirth R, Leonard EJ, Lim B. Phase 2 safety and efficacy results of TAK-228 in combination with exemestane or fulvestrant in postmenopausal women with ER-positive/HER2-negative metastatic breast cancer previously treated with everolimus [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD1-09.

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.

11528 Background: Chondrosarcoma is one of the most common bone malignancies in adults, and the third most common in pediatric patients (pts). The most prevalent subtype, conventional chondrosarcoma, is a slow growing tumor that is historically known to be refractory to chemotherapy. Anecdotal reports indicated a role for anti-PD-(L)1 in the treatment of this disease. This is the first prospective report on the efficacy of the PD-L1-targeting agent, atezolizumab, in this rare disease. Methods: Patients (pts) ages 2 and older with unresectable grade 2 or 3 conventional chondrosarcoma were eligible. No prior anti-PD-(L)1 treatment was allowed, otherwise pts were eligible irrespective of prior therapies as long as protocol-specified washout period requirements were met. Pts received atezolizumab 1200 mg (15 mg/kg with 1200 mg cap in pediatric pts) once every 21 days. Imaging was carried out at end of cycle 3, and then every two cycles. Research biopsies were collected from adult pts prior to C1D1, prior to C3D1, and at progression. Immuno-pharmacodynamic (IO-PD) studies were performed on paired tumor samples and circulating immune cells to help elucidate signaling pathways mediating the immune response, with focus on subsets of effector cells in the tumor microenvironment. Results: A total of 9 pts (7 males, 2 females) were enrolled in 6 centers across the US and Canada. Six pts were Caucasian/White, 1 Asian, 1 Hispanic, and 1 unknown. Median age was 49 years (42-72). No objective responses were seen. Three pts (33%) experienced disease stability (SD) per RECIST 1.1, for a median duration of 21 weeks as of data cutoff (January 2022). A patient with SD remains on active treatment (tx) for 35 weeks. Three patients had no tx-related adverse events (AEs). Six pts (67%) experienced at least one tx-related AE. Two patients experienced > G2 AEs, but only one was considered tx-related (lymphopenia). Immune-related AEs were all G1/2 and included hepatitis (2), hypothyroidism (1), hyperthyroidism (1), and maculopapular rash (1). IO-PD studies are ongoing and will be reported at the conference if available. Conclusions: Atezolizumab was well-tolerated but demonstrated limited activity in this cohort of pts with few treatment options. Ongoing IO-PD studies will provide insight into atezolizumab’s effect upon immune cell content and activation in the tumor microenvironment that will help design future immunotherapy trials in this disease and other sarcoma types. The study was funded by NCI Contract HHSN261201500003I. Clinical trial information: NCT04458922.

Purpose:This open-label, multicenter, phase IB/II study evaluated sapanisertib, a dual inhibitor of mTOR kinase complexes 1/2, plus exemestane or fulvestrant in postmenopausal women with hormone receptor–positive (HR+)/HER2-negative (HER2−) advanced/metastatic breast cancer.Patients and Methods:Eligible patients had previously progressed on everolimus with exemestane/fulvestrant and received ≤3 (phase IB) or ≤1 (phase II) prior chemotherapy regimens. Patients received sapanisertib 3 to 5 mg every day (phase IB), or 4 mg every day (phase II) with exemestane 25 mg every day or fulvestrant 500 mg monthly in 28-day cycles. Phase II enrolled parallel cohorts based on prior response to everolimus. The primary objective of phase II was to evaluate antitumor activity by clinical benefit rate at 16 weeks (CBR-16).Results:Overall, 118 patients enrolled in phase IB (n = 24) and II (n = 94). Five patients in phase IB experienced dose-limiting toxicities, at sapanisertib doses of 5 mg every day (n = 4) and 4 mg every day (n = 1); sapanisertib 4 mg every day was the MTD in combination with exemestane or fulvestrant. In phase II, in everolimus-sensitive versus everolimus-resistant cohorts, CBR-16 was 45% versus 23%, and overall response rate was 8% versus 2%, respectively. The most common adverse events were nausea (52%), fatigue (47%), diarrhea (37%), and hyperglycemia (33%); rash occurred in 17% of patients. Molecular analysis suggested positive association between AKT1 mutation status and best treatment response (complete + partial response; P = 0.0262).Conclusions:Sapanisertib plus exemestane or fulvestrant was well tolerated and exhibited clinical benefit in postmenopausal women with pretreated everolimus-sensitive or everolimus-resistant breast cancer.

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 Icrucumab (ICR) and ramucirumab (RAM) bind vascular endothelial growth factor (VEGF) receptors 1 and 2 (VEGFR-1 and -2), respectively. This open-label, randomized phase II study evaluated their efficacy and safety in combination with capecitabine (CAP) in patients with previously treated unresectable, locally advanced or metastatic breast cancer. Methods Patients were randomly assigned (1:1:1) to receive CAP (1,000 mg/m2 orally twice daily, days 1-14) alone or in combination with RAM (10 mg/kg intravenously [IV], days 1 and 8) (RAM + CAP) or ICR (12 mg/kg IV, days 1 and 8) (ICR + CAP) every 21 days. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), tumor response, safety, and pharmacokinetics. Results Of 153 patients randomized, 150 received treatment. Median PFS (95% confidence interval) was 22.1 (12.1-36.1) weeks on RAM + CAP, 7.3 (6.3-13.0) weeks on ICR + CAP, and 19.0 (12.1-24.3) weeks on CAP (hazard ratios [HRs]: 0.691, p = .1315, RAM + CAP versus CAP; 1.480, p = .0851, ICR + CAP versus CAP). Median OS was 67.4 weeks on RAM + CAP, 62.1 weeks on ICR + CAP, and 71.6 weeks on CAP (HRs: 1.833, p = .0283, RAM + CAP versus CAP; 1.468, p = .1550, ICR + CAP versus CAP). There was no statistically significant difference in PFS or OS between either combination arm and CAP. Treatment-related adverse events more frequent (by ≥10%) on RAM + CAP than on CAP were constipation, decreased appetite, headache, epistaxis, and hypertension. Those more frequent (by ≥10%) on ICR + CAP than CAP were anemia, increased lacrimation, periorbital edema, nausea, vomiting, peripheral edema, facial edema, dehydration, and dyspnea. Conclusion Combining RAM or ICR with CAP did not improve PFS in the targeted study population. The Oncologist 2017;22:245-254 IMPLICATIONS FOR PRACTICE: Icrucumab and ramucirumab are recombinant human IgG1 monoclonal antibodies that bind vascular endothelial growth factor (VEGF) receptors 1 and 2 (VEGFR-1 and -2), respectively. VEGFR-1 activation on endothelial and tumor cell surfaces increases tumor vascularization and growth and supports tumor growth via multiple mechanisms, including contributions to angiogenesis and direct promotion of cancer cell proliferation. Strong preclinical and clinical evidence suggests key roles for VEGF and angiogenesis in breast cancer growth, invasion, and metastasis. This randomized phase II study evaluated the efficacy and safety of each antibody in combination with capecitabine in patients with previously treated unresectable, locally advanced or metastatic breast cancer.

Aggressive cancer cells are characterized by their capacity to proliferate indefinitely and to propagate a heterogeneous tumor comprised of subpopulations with varying degrees of metastatic propensity and drug resistance properties. Particularly daunting is the challenge we face in the field of oncology of effectively targeting heterogeneous tumor cells expressing a variety of markers, especially those associated with a stem cell phenotype. This dilemma is especially relevant in breast cancer, where therapy is based on traditional classification schemes, including histological criteria, differentiation status, and classical receptor markers. However, not all patients respond in a similar manner to standard-of-care therapy, thereby necessitating the need to identify and evaluate novel biomarkers associated with the difficult-to-target stem cell phenotype and drug resistance. Findings related to the convergence of embryonic and tumorigenic signaling pathways have identified the embryonic morphogen Nodal as a promising new oncofetal target that is reactivated only in aggressive cancers, but not in normal tissues. The work presented in this paper confirms previous studies demonstrating the importance of Nodal as a cancer stem cell molecule associated with aggressive breast cancer, and advances the field by providing new findings showing that Nodal is not targeted by standard-of-care therapy in breast cancer patients. Most noteworthy is the linkage found between Nodal expression and the drug resistance marker ATP-binding cassette member 1 (ABCA1), which may provide new insights into developing combinatorial approaches to overcome drug resistance and disease recurrence.

Neoadjuvant chemotherapy (NAC) is a significant modality in breast cancer therapy. We sought to characterize prognostic factors in patients scheduled for NAC who had a pretreatment positron-emission tomography paired with diagnostic quality contrast-enhanced computed tomography (CT) (positron-emission tomography/CT [PET/CT]). A total of 118 breast cancer patients were analyzed through chart review who underwent pretreatment PET/CT imaging and received NAC from 2008 to 2014. We collected information on molecular markers, PET/CT, pathologic complete response (pCR), survival, and disease status. Pretreatment standard uptake value (SUV) max of the primary breast tumor showed no relationship to pCR; however, there was a statistically significant relationship with relapse-free survival (RFS) using univariate cox regression (P = 0.03, odds ratio (OR) = 1.06 [1.01–1.12]) with comparable findings observed with overall survival (OS). Multivariate analysis revealed SUV max to be significantly correlated with shortened OS (P = 0.022, OR = 1.08 [1.01–1.16]), with a similar trend reported for RFS. By pathological subtype, this correlation was the strongest within hormone receptor (HR+)/human epidermal growth factor receptor 2 (HER2−) tumors. In addition, Kaplan–Meier estimates demonstrated a significant difference between the RFS of triple-negative tumors and HER2 positive tumors (P = 0.001). Interestingly, within this cohort, multivariate Cox regression analysis showed HER2 positivity to be associated with favorable outcome (P = 0.04, HR = 0.22 [0.05–0.94]). These findings demonstrate a significant association between SUV max of HR+/HER2−− tumors and relapse-free and OS. Furthermore, highlighted here is the favorable survival in the once classically aggressive HER2+ breast cancer subgroup.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809–22. ©2016 AACR.

Tumor cells inherit from their normal precursors an extensive stress response machinery that is critical for survival in response to challenges including oxidative stress, wounding, and shear stress. Kruppel-like transcription factors, including KLF4 and KLF5, are rarely affected by genetic alteration during tumorigenesis, but compose key components of the stress response machinery in normal and tumor cells and interact with critical survival pathways, including RAS, p53, survivin, and the BCL2 family of cell death regulators. Within tumor cells, KLF4 and KLF5 play key roles in tumor cell fate, regulating cell proliferation, cell survival, and the tumor-initiating properties of cancer stem–like cells. These factors can be preferentially expressed in embryonic stem cells or cancer stem–like cells. Indeed, specific KLFs represent key components of a cross-regulating pluripotency network in embryonic stem cells and induce pluripotency when coexpressed in adult cells with other Yamanaka factors. Suggesting analogies between this pluripotency network and the cancer cell adaptive reprogramming that occurs in response to targeted therapy, recent studies link KLF4 and KLF5 to adaptive prosurvival signaling responses induced by HER2-targeted therapy. We review literature supporting KLFs as shared mechanisms in stress adaptation and cellular reprogramming and address the therapeutic implications. Cancer Res; 76(7); 1677–82. ©2016 AACR.

Background: In this phase Ib study, the activity of T-DM1 plus N was assessed in patients (pt) previously treated with trastuzumab, pertuzumab, and a taxane (H+P+T). Several mechanisms of resistance have been hypothesized in pts progressing following H+P+T, including acquired alterations in the ERBB (HER) family proteins, reactivation of bypass or parallel pathways, or selective elimination of HER2-overexpressing clones. Loss of HER2 amp has been shown to occur in 25-35% of pts with residual tumor after neoadjuvant therapy or in metastatic disease after initial therapy with chemotherapy and HER2-targeted agents. Data on concordance of HER2 status between tissue and blood is limited. In 7 pts with cfDNA HER2 amp, concomitant tissue was concordant in all 7 pairs and response to anti-HER2 therapy occurred in 6. In our study we have retrospectively analyzed cfDNA in blood samples obtained at study entry. Methods: Eligible pts had prior H+P+T as neoadjuvant therapy, or 1st-line metastatic disease, measurable disease, ECOG PS ≤2, and adequate hematologic, renal, and liver function. Pts with stable brain metastases were eligible. Treatment consisted of T-DM1 3.6 mg/kg iv q3wk and N 120, 160, 200, or 240 mg/d using a 3+3 dose-escalation design. HER2+ was determined at initial diagnosis; tissue confirmation at study entry (after H+P+T progression) was not required. Blood was collected in for pharmacokinetic analyses of N peak and trough, and for cfDNA using the Guardant360 assay, which is a 73-gene next-generation cfDNA-sequencing panel that detects SNVs, indels, CNAs, and fusions, utilizing Digital Sequencing and custom bioinformatics methods for error correction. The cut-off for HER2 amp was a copy number of ≥2.0 established by Guardant based on training-set data. Results: There were 27 H+P+T-resistant pts enrolled and all pts had a blood sample analyzed for HER2 amp. Eighteen pts were evaluable for efficacy at 6 wks and 11 pts at 12 wks. Dose-limiting toxicity occurred in 6 pts during cycle 1, 1 pt was withdrawn for non-compliance, and 2 pts were withdrawn for disease complications. The recommended phase II dose of N was determined to be 160 mg/d. Responses were seen at all dose-levels of N. Pharmacokinetic analyses did not show a clear relationship with either peak or trough and dose-level. Ten pts showed HER2 amp in blood and 17 were non-amp. Of 18 pts evaluable after 2 cycles (6 wks), 12 pts had an objective response (7 amp; 5 non-amp) and 5 had progressive disease (1 amp; 4 non-amp). At 12 wks, there were 3 CRs and 8 PRs (7 amp; 4 non-amp). All CRs were in amp pts and lasted 364, 510, and 859+ days. Conclusions: HER2 amp as determined by cfDNA was found in 10 of 27 pts. The deeper and more prolonged (>12 wk) responses occurred in 7 of 10 amp HER2 pts v 4 of 17 non-amp HER2 pts (p=0.04). In our ongoing phase II study of this regimen concomitant tissue and blood will be analyzed to better understand potential benefit or lack of benefit, with continued use of anti-HER2 therapy after progression on anti-HER2 therapies. Support: Puma Biotechnology, Inc. Citation Format: Abraham J, Puhalla SL, Sikov WM, Montero AJ, Salkeni MA, Razaq WA, Beumer JH, Kiesel BF, Buyse ME, Adamson LM, Srinivasan A, Pogue-Geile KL, Allegra CJ, Nagy RJ, Jacobs SA. Analysis of ERBB2 (HER2) amplification by ctDNA in a phase Ib dose-escalation trial evaluating trastuzumab emtansine (T-DM1) with neratinib in women with metastatic disease with initially diagnosed HER2+ breast cancer: NSABP FB-10 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-04.

200 Background: Real-world practice patterns often differ from treatment in clinical trials. We assessed real-world standard-of-care treatment with palbociclib (PAL) in the context of previously reported PALOMA trial results. Methods: POLARIS is a prospective, observational study of PAL in patients (pts) with hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Baseline demographics, clinical characteristics, initial treatment dose, dose modifications, dose delays, and adverse events (AEs) during the first 6 months (mo) of treatment were analyzed. Results: 412 pts enrolled at 92 US sites had at least 6 mo of PAL treatment; 73% received PAL in the first-line setting and 27% in second or later line. While a majority of pts received the recommended dose of 125 mg/d; 6% and 1% started at 100 mg and 75 mg, respectively. Physician-reported reasons for choosing a lower dose were: comorbidities (31%), pt age (21%), past treatment (10%), patient preference (3%), and other (34%). Selected pt and clinical characteristics, dosing, and treatment outcomes in POLARIS and in PALOMA-2 are shown in Table. Conclusions: In a real-world data set of 6 mo of PAL treatment, most pts started at the recommended dose, with tolerability and safety outcomes consistent with those reported in clinical trials. Differing populations, treatment patterns, and outcome reporting in real world vs trial settings underscore the need to study real-world practices and outcomes. Clinical trial information: NCT03280303 [Table: see text]

192 Background: Adverse effects of endocrine therapy (ET), especially musculoskeletal symptoms (MSS), represent the major cause of treatment discontinuation in breast cancer (BC) patients, resulting in increased risk of recurrence. Aromatase inhibitors (AI), the mainstay of ET, are associated with a high incidence of MSS such as arthralgia and myalgia. The hypothesis of whether switching ET subclass improves adherence and symptoms was tested. Methods: We conducted a retrospective single site chart review of female patients with nonmetastatic BC, who were seen at Cancer Clinic from January to June of 2017. All patients received AI as a first choice of adjuvant ET; those with disease recurrence while on ET were excluded. We collected data on baseline health, adherence and discontinuation rate, as well as whether switching to a different ET agent (another AI or tamoxifen) was attempted. The subsequent compliance and symptomatology after switching were assessed. Results: 483 eligible patients were identified and divided into 4 groups based upon adherence: 354 (73.3%) were found to be compliant with AI without interruption, 109 (22.5%) switched to a different ET and remained compliant (≥ 1 switch), 16 (3.3%) eventually discontinued ET despite attempting ≥ 1 switch, and 5 patients (1.0%) stopped treatment without a switch. On comparison of the different groups, patients who permanently discontinued ET were more likely to be elderly and have preexisting musculoskeletal condition. MSS was found to be the single most common cause for AI intolerance (105 patients, 81.4%). The median time to switch was 6.3 months (0.3-63.9). Among the compliant switchers, most needed only 1 switch (79) but there were patients who required 2 or 3 switches (23 and 6, respectively). Majority of patients switched from anastrozole to exemestane (84, 51.8%). Significant improvement in symptoms after the switch was reported by 73.3% of patients. Conclusions: Switching between different ET subclasses is an effective treatment strategy to alleviate side effects and enhance adherence to ET. A higher compliance rate of 95.8% was observed in our study compared to a national average estimated at < 80%.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens including surgical resection, radiation and chemotherapy. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) found in about a third of GBMs. It confers enhanced tumorigenic behavior and is associated with chemo- and radio-resistance. GBM patients testing positive for EGFRvIII have a bleaker prognosis than those who do not. Targeting EGFRvIII positive tumors via vaccines or antibody-drug-conjugates represents a new challenging therapeutic avenue with potential great clinical benefits. In this study, we developed a strategy to detect EGFRvIII deletion in the circulating tumor DNA. The overall goal is to identify a simple and robust biomarker in the peripheral blood of patients diagnosed with GBM in order to follow their disease status while on treatment. Thirteen patients were included in this study, three of which were found to carry the EGFRvIII deletion. The circulating DNA status for EGFRvIII correlates with the analysis performed on the respective tumor samples, and its level seems to correlate with the extent of the tumor resection. This semi-quantitative blood biomarker may represent a strategy to (1) screen patients for an anti-EGFRvIII therapy and (2) monitor the patients' response to treatment.

1027Background: T-DM1 is an antibody-drug conjugate composed of trastuzumab and the maytansinoid antimicrotubule, DM1. T-DM1 was granted FDA approval in 2nd-line MBC after prior trastuzumab (T) and...

e23051 Background: The latest ASCO guidelines regarding tumor markers in breast cancer do not recommend routine monitoring of serum CA 15-3 (CA) levels alone as a marker for relapse. They do, however, acknowledge their use in conjunction with a patient’s history, physical exam, and diagnostic imaging. The study objective is to evaluate CA as a marker for relapse and determine its continued use in our patient population. Methods: We performed a retrospective analysis on female stage I-III breast cancer patients with an elevated CA marker treated in our cancer center between 2009-2014. Patients with metachronous or synchronous malignancies were excluded. Patients were categorized into three groups: Group 1 (elevated CA at relapse), Group 2 (normal CA at relapse, with elevation post-relapse), Group 3 (elevated CA without relapse). Categorical variables were collected to fulfill our objectives and the Fisher’s exact test was used to assess the correlation between them. The incidence rate and its 95% confidence interval were estimated based on the binomial distribution. Results: Out of 340 initially screened patients, 92 met our inclusion criteria: Group 1 (n = 25), Group 2 (n = 23), Group 3 (n = 44). The PPV for an elevated CA as a marker for relapse was 36% (95% CI: 26-48%). On routine surveillance, patients with elevated CA levels were more likely to have relapse if they presented with nausea (p = 0.02), myalgia (p = 0.003), or axial bone pain (p = 0.04). At relapse, an elevated CA was associated with fatigue (p = 0.02), myalgia (p = 0.01), liver metastases (p = 0.01), axial bone metastases (p = 0.005), and peripheral bone metastases (p = 0.0002). In patients with an elevated CA, a BMI < 25 had a higher incidence of relapse in comparison to those with a BMI ≥ 25 (p = 0.01). Conclusions: Our study suggests that a patient’s history, physical, and symptom-dictated imaging should be the main way to screen for relapse in stage I-III breast cancer. Based on the higher incidence of relapse in patients with both an elevated CA and symptoms of nausea, myalgia, or axial bone pain, we conclude that serum CA levels may be used as an adjunctive test in symptomatic patients. Our data also suggests that elevated CA levels may be less useful in detecting relapse in patients with BMI ≥ 25.

e12113 Background: Neoadjuvant chemotherapy (NAC) is an important modality in breast cancer treatment. We sought to identify pre-treatment prognostic factors in patients who had positron emission tomography paired with diagnostic quality contrast-enhanced CT (PET/CT) prior to neoadjuvant chemotherapy with respect to pathologic complete response (pCR) , survival and relapse-free survival (RFS). Methods: We retrospectively analyzed 118 breast cancer patients who had pre-treatment PET/CT imaging and received NAC from 2008-2014. We collected data on molecular markers, PET/CT, pCR, survival, and disease status. Results: The median follow up was 44 months(range 7.3-101.5),median age was 51 years; 47% were stage II, 53% stage III. 52% of patients had hormone receptor (HR) positive/HER2 negative disease, 31% of tumors were HER2 positive, and 17% of tumors were triple-negative. 92.5% with HER2 positive tumors received NAC containing at least one HER2 targeted agent. Pre-treatment standard uptake value (SUV) max of the primary breast tumor showed no statistically significant relationship to survival, RFS, or pCR. PET avid (>2 SUV) extra-axillary nodes such as internal mammary and supraclavicular was associated with a non-statistically significant trend towards reduced RFS (p=0.06, HR=0.13-1.06). pCR overall was 37.5% for HER2 positive tumors, 15% in triple-negative tumors, and 8% in HR positive/HER2 negative tumors. Log-rank analysis with post-hoc pairwise comparisons showed a significant difference between the RFS of triple-negative tumors and HER2 positive tumors (p=0.001), while comparison between HR positive/HER2 negative and HER2 positive was not statistically significant (p=0.11). Multivariate cox regression analysis, which included grade and stage of tumors, showed HER2 positivity to be associated with a favorable outcome (p=0.04, HR=0.22 (0.05-0.94)). Conclusions: Within this cohort, pre-treatment SUV max of the primary tumor showed no prognostic value with regard pCR or RFS. PET avid extra-axillary metastasis trended towards reduced RFS. Patients with HER2 positive tumors had the highest pCR and RFS comparable to classically favorable subgroups such as HR positive/HER 2 negative.

Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS–extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells.

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor in adults. It is a highly vascularized tumor, and the advent of angiogenic inhibitors, in particular bevacizumab, is thought to be promising for the treatment of these tumors. However, bevacizumab has been associated with an increased risk of arterial and venous thromboembolism and hemorrhage. We report a case of superior vena cava syndrome developing in a glioblastoma patient treated with bevacizumab. Superior vena cava thrombosis in the setting of treatment with vascular endothelial growth factor has not been well-described. The issue arises as how to best manage the hemostatic complications of antiangiogenic agents in patients who have an established risk of bleeding and thrombosis with their GBM. This report discusses the individual risk of bleeding and thrombosis associated with GBM and the use of bevacizumab. Studies suggest that GBM patients who require therapeutic anticoagulation for venous thromboembolic complications while on bevacizumab therapy can be anticoagulated with the risk of bleeding being comparable to that expected from therapeutic anticoagulation alone. However, the potential risks and benefits should be thoroughly discussed with each patient before starting anticoagulation therapy for thrombotic complications.

10078Background: Women undergoing mastectomy worry about effects on upper extremity function. Past research has shown long-standing problems with stiffness, lymphedema or weakness that impairs func...

P-glycoprotein (P-gp) is a brain-to-blood efflux system that controls the ability of many drugs and endogenous substances to access the brain. In vitro work has shown that inflammatory states mediated through lipopolysaccharide (LPS) and tumor necrosis factor-alpha first impair and then stimulate P-gp activity. Here, we determined whether LPS can affect P-gp function in vivo. Mice treated with a single intraperitoneal injection of LPS (3 mg/kg) showed an inhibition of P-gp function. As assessed by brain perfusion, inhibition began 18 h after LPS administration and lasted until 36 h after administration. P-gp protein was increased by 44%, consistent with P-gp inhibition occurring through post-translational mechanisms. Unlike other effects of LPS on blood–brain barrier function, neither nitric oxide nor prostaglandin inhibition had an effect. We conclude that induction of proinflammatory states as exemplified by LPS treatment can inhibit P-gp function in vivo at the blood–brain barrier.

The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

Background/Purpose: The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor, BEZ235, was shown to be efficacious as an inhibitor of cancer cell proliferation in combination with the mTOR-allosteric inhibitor, RAD001 or everolimus, in a preclinical model over everolimus alone. We hypothesized that the combinatorial effect on the mTOR and PI3K pathway would result in a synergistic anti-tumor effect in humans. Therefore, we designed a first in human, phase 1b trial, in order to study the safety and pharmacokinetics of the combination of BEZ235 and everolimus in patients with solid tumor malignancies. Methods: Nineteen patients with solid tumor malignancies that had progressed on standard of care treatment were enrolled. The experimental medication, BEZ235, was provided to us by Novartis. BEZ235 was administered orally once a day at escalating doses of 200, 400, and 800mg and everolimus was administered orally once a day at 2.5mg in 28 day cycles. Results: The most common toxicities observed included fatigue (68%), diarrhea (74%), nausea (63%), mucositis (42%) and elevated liver enzymes (63%). The maximum tolerated dose (MTD) was not reached due to toxicity resulting in early discontinuation of the trial. BEZ235 serum levels were inconsistent suggesting poor absorption. Everolimus exposure was increased over what would be expected for administration alone, likely due to the previously postulated drug interaction between BEZ235 and everolimus. Of the twelve patients evaluated for response, one had SD at 5 weeks and no PR or CR was observed. Conclusion: The combination therapy of BEZ235 and everolimus was shown to be poorly tolerated in our patient population likely due to poor absorption, drug interaction, and inter-patient variability leading to inconsistent drug levels. Although, there were no confirmed responses, one patient did respond clinically for a brief period. We expect that the combination of everolimus with new dual inhibitors with improved toxicity profiles and increased absorption will be more promising.

e13023 Background: Several strategies have been recently developed to target EGFRvIII in Glioblastoma Multiforme (GBM), including vaccines (CDX-110) and antibody-drug conjugates (AMG595) and represents a new challenging therapeutic avenue with potential great clinical benefits. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) found in about a third of GBMs, confers enhanced tumorigenic behavior and is associated with chemo- and radio-resistance. GBM patients testing positive for EGFRvIII have a bleaker prognosis than those who do not. Methods: We developed a strategy to detect EGFRvIII deletion in the circulating tumor DNA. The purpose of this study is to identify a simple and robust biomarker from the peripheral of patients diagnosed with GBM in order to screen patients for the EGFRvIII deletion. 11 patients have been included in this study. Results: The circulating DNA status for EGFvIII correlates with the analysis performed on the respective tumors samples, and its level seems to be correlated with the extend of the tumor resection. Conclusions: This semi-quantitative blood biomarker may represents a strategy to 1) screen patients for anti-EGFRvIII therapy 2) monitor the therapeutic response to specific targeted therapies.

e13518 Background: The mammalian target of rapamycin (mTOR) is a critical signaling pathway in many tumors including cancers of the breast and colon, glioblastoma multiforme, and hepatocellular carcinoma (HCC). Preclinical studies demonstrated the combination of BEZ235, a competitive dual phosphatidylinositide 3-kinase (PI3K)/mTOR inhibitor, and the mTOR inhibitor, everolimus led to greater regression of a carcinogen-induced HCC than treatment with higher doses of either drug alone. Based on this, we initiated a study of BEZ235 combined with everolimus in patients with advanced solid tumors. Methods: A single institution phase Ib dose-escalation study. Patients with advanced solid malignancies, no available standard of care treatment option, and ECOG performance status 0-2 were eligible. Prior treatment with PI3K inhibitors was not allowed. Sequential cohorts of 3-6 patients were treated. The starting dose was everolimus 2.5 mg and BEZ235 200 mg daily in an oral sachet formulation. Cohort 2 received everolimus 2.5 mg and BEZ235 400 mg daily. Pharmacokinetic and pharmacodynamic studies were performed during the first cycle. The phosphorylation of specific downstream effectors of the mTOR pathway was assessed in peripheral blood mononuclear cells (PBMC). Results: Eleven patients, median age 58 (36-73 years) were treated. Tumors included non-small cell lung cancer, colon cancer, and glioblastoma, hepatocellular carcinoma, pancreatic cancer, esophageal cancer, adenoid cystic carcinoma of the larynx, and appendiceal carcinoma. Four patients were treated on cohort 1. None experienced dose-limiting toxicity. Seven patients were treated on the second dose cohort. One patient withdrew necessitating replacement and another developed grade 3 stomatitis from herpes virus requiring cohort expansion. The most common adverse events were thrombocytopenia, lymphopenia, transaminitis, diarrhea, nausea and fatigue. No tumor responses were noted. PBMC showed a decrease in 4E-BP1S65 phosphorylation on day 28 in 2 of 3 patients in cohort 1. Conclusions: The combination of BEZ235 and everolimus was well tolerated at these doses. The trial remains open to accrual. Clinical trial information: NCT01508104.

TPS3116 Background: Downregulation of a number of signaling pathways, including the mammalian target of rapamycin (mTOR) pathway, has been demonstrated to be efficacious in a large range of solid tumors such as breast, colon, endometrial, glial and hepatocellular carcinoma (HCC). However, we find that rapamycins lead to suppression of a negative feedback loop from S6 Kinase 1 (S6K1) to Protein Kinase B (PKB), leading to hyperactivation of PKB. In pre-clinical studies using a mouse model of carcinogen-induced HCC, we have demonstrated that combining BEZ235 (a potent and highly selective reversible ATP site competitive inhibitor of PI3K and mTOR) with everolimus (an allosteric inhibitor of mTOR) synergizes to inhibit tumor growth. BEZ235, an orally administered agent, has demonstrated preliminary antitumor activity in a first-in-human phase I study. The current study will evaluate this combination in patients with a variety of solid malignancies that includes glioblastoma multiforme (GBM). Methods: This study is divided into a phase 1b portion designed to determine safety of increasing doses of the combination, with extensive pharmacokinetics, pharmacodynamics and pharmacogenomics analysis; and a phase 2 portion that includes both solid tumors and GBM based on predominance of the mTOR and PI3K deregulation in these tumors, to determine preliminary anti-tumor activity and the recommended dose for phase 2 studies. We will also integrate biomarker assessment for gene expression products of the mTOR downstream pathway such as eukaryotic initiation factor 4E binding protein (4EBP1) and S6 kinase (S6K). The phase 1b portion has started accruing.

e16017 Background: Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer, but no predictive biomarkers of cetuximab activity have yet been identified. Methods: We report on a patient with HNSCC who had a complete tumor regression following treatment with cetuximab given as a single agent after initial surgery and radiation therapy. The EGFR protein expression level, gene copy number and sequence were assessed from both normal and tumor tissues from this patient. Results: Besides protein overexpression and gene amplification in the tumor tissue, sequencing of the EGFR gene from the patient revealed the presence of two somatic mutations, one in the kinase domain (R705G) and the other in the ligand binding domain (P546S). Cells that stably express these EGFR mutants were treated with cetuximab and their sensitivity to the drug was compared to cells expressing the wildtype gene. While the P546S mutation sensitized NIH-3T3 cells to cetuximab, R705G had a marginal effect. The double mutant (P546S/R705G) behaved the same as the P546S mutant, indicating that the mutation in the kinase domain does not contribute to the increased sensitivity to cetuximab. No mutations were found in K-RAS or B-RAF genes and there was no indication that the tumor was HPV-positive. Conclusions: Our results support a role for the P546S mutation in cetuximab sensitivity. To our knowledge, this is the first report of a somatic mutation in the EGFR ligand binding domain that may contribute to increased sensitivity to cetuximab. Other factors including EGFR copy number, EGFR over-expression and the immune response, as indicated by a very adverse side effect that correlated with the antitumor activity, may have also contributed to the observed response. It remains to be determined how frequently this mutation occurs in patients with HNSCCs and other cancers. Prospective evaluation of cetuximab anti-tumor activity in patients harboring P546S mutation needs to be clinically evaluated.

Background The combination of everolimus and the imidazoquinoline derivative, BEZ235 (dactolisib), a dual PI3K/mTOR inhibitor, demonstrated synergy in a preclinical model. Objective To establish clinical feasibility, a phase Ib dose-escalation trial investigating safety and pharmacokinetics of this combination in patients with advanced tumors was performed. Patients and Methods BEZ235 was orally administered daily in escalating doses of 200, 400, and 800 mg along with everolimus at 2.5 mg daily in 28-day cycles. Nineteen patients were enrolled. Adverse events and tumor responses were evaluated using CTCAE v4.0 and RECIST 1.1, respectively. Pharmacokinetic analyses were performed. Results Common toxicities observed included fatigue, diarrhea, nausea, mucositis, and elevated liver enzymes. No confirmed responses were observed. BEZ235 pharmacokinetics exhibited dose-proportional increases in Cmax and AUC0-24 over the three doses, with high inter-individual variability. Non-compartmental and population pharmacokinetic-based simulations indicated significant increases in everolimus Cmax and AUC0-24 on day 28 and decreased clearance to 13.41 L/hr. Conclusions The combination of BEZ235 and everolimus demonstrated limited efficacy and tolerance. BEZ235 systemic exposure increased in a dose-proportional manner while oral bioavailability was quite low, which may be related to gastrointestinal-specific toxicity. The changes in steady-state pharmacokinetics of everolimus with BEZ235 highlight potential drug–drug interactions when these two drugs are administered together. Clinicaltrials.gov: NCT01508104 Electronic supplementary material The online version of this article (doi:10.1007/s11523-017-0482-9) contains supplementary material, which is available to authorized users.