NSABP FB-10: a phase Ib/II trial evaluating ado-trastuzumab emtansine (T-DM1) with neratinib in women with metastatic HER2-positive breast cancer.

BACKGROUND: We previously reported our phase Ib trial, testing the safety, tolerability, and efficacy of T-DM1 + neratinib in HER2-positive metastatic breast cancer patients. Patients with ERBB2 amplification in ctDNA had deeper and more durable responses. This study extends these observations with in-depth analysis of molecular markers and mechanisms of resistance in additional patients. METHODS: Forty-nine HER2-positive patients (determined locally) who progressed on-treatment with trastuzumab + pertuzumab were enrolled in this phase Ib/II study. Mutations and HER2 amplifications were assessed in ctDNA before (C1D1) and on-treatment (C2D1) with the Guardant360 assay. Archived tissue (TP0) and study entry biopsies (TP1) were assayed for whole transcriptome, HER2 copy number, and mutations, with Ampli-Seq, and centrally for HER2 with CLIA assays. Patient responses were assessed with RECIST v1.1, and Molecular Response with the Guardant360 Response algorithm. RESULTS: The ORR in phase II was 7/22 (32%), which included all patients who had at least one dose of study therapy. In phase I, the ORR was 12/19 (63%), which included only patients who were considered evaluable, having received their first scan at 6 weeks. Central confirmation of HER2-positivity was found in 83% (30/36) of the TP0 samples. HER2-amplified ctDNA was found at C1D1 in 48% (20/42) of samples. Patients with ctHER2-amp versus non-amplified HER2 ctDNA determined in C1D1 ctDNA had a longer median progression-free survival (PFS): 480 days versus 60 days (P = 0.015). Molecular Response scores were significantly associated with both PFS (HR 0.28, 0.09-0.90, P = 0.033) and best response (P = 0.037). All five of the patients with ctHER2-amp at C1D1 who had undetectable ctDNA after study therapy had an objective response. Patients whose ctHER2-amp decreased on-treatment had better outcomes than patients whose ctHER2-amp remained unchanged. HER2 RNA levels show a correlation to HER2 CLIA IHC status and were significantly higher in patients with clinically documented responses compared to patients with progressive disease (P = 0.03). CONCLUSIONS: The following biomarkers were associated with better outcomes for patients treated with T-DM1 + neratinib: (1) ctHER2-amp (C1D1) or in TP1; (2) Molecular Response scores; (3) loss of detectable ctDNA; (4) RNA levels of HER2; and (5) on-treatment loss of detectable ctHER2-amp. HER2 transcriptional and IHC/FISH status identify HER2-low cases (IHC 1+ or IHC 2+ and FISH negative) in these heavily anti-HER2 treated patients. Due to the small number of patients and samples in this study, the associations we have shown are for hypothesis generation only and remain to be validated in future studies. Clinical Trials registration NCT02236000.

Sapanisertib Plus Exemestane or Fulvestrant in Women with Hormone Receptor-Positive/HER2-Negative Advanced or Metastatic Breast Cancer.

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.

Resistance to Immunotherapy: Mechanisms and Means for Overcoming.

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.

The Stem Cell Phenotype of Aggressive Breast Cancer Cells.

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.

A Phase Ib Study of the Dual PI3K/mTOR Inhibitor Dactolisib (BEZ235) Combined with Everolimus in Patients with Advanced Solid Malignancies.

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.

Randomized Phase II Study of Ramucirumab or Icrucumab in Combination with Capecitabine in Patients with Previously Treated Locally Advanced or Metastatic Breast Cancer.

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.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer.

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.

Kruppel-like Pluripotency Factors as Modulators of Cancer Cell Therapeutic Responses.

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.

MicroRNAs 206 and 21 cooperate to promote RAS-extracellular signal-regulated kinase signaling by suppressing the translation of RASA1 and SPRED1.

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.

Detection of EGFRvIII mutant DNA in the peripheral blood of brain tumor patients.

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.

Lipopolysaccharide impairs blood-brain barrier P-glycoprotein function in mice through prostaglandin- and nitric oxide-independent pathways.

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.