A Phase Ib Study of Alpelisib (BYL719), a PI3Kα-specific Inhibitor, with Letrozole in ER+/HER2-Negative Metastatic Breast Cancer

VSports注册入口 - Patient Population

The patient population included post-menopausal patients with histologically confirmed ER+/HER2− metastatic breast cancer refractory to at least one line of endocrine therapy in the metastatic setting, or diagnosed with metastatic breast cancer during or within 1 year of adjuvant endocrine therapy; evaluable disease as defined by Response Evaluation Criteria In Solid Tumors (RECIST); age ≥18 years; life expectancy ≥6 months; ECOG performance status ≤1; adequate bone marrow, hepatic, and renal function; and fasting plasma glucose levels ≤140 mg/dL (7.8 mmol/L). A tumor specimen (primary or metastatic) from archival material or a fresh biopsy was required for study enrollment. Key exclusion criteria were CYP3A4 modifier drug treatment ≤2 weeks before starting alpelisib, clinically-manifested diabetes mellitus, and prior treatment with PI3K inhibitors. Prior treatment with everolimus was allowed.

Approval was obtained from the ethics committees (IRB #101057, Vanderbilt University) at the participating institutions and regulatory authorities. All patients gave informed consent. The study followed the Declaration of Helsinki and Good Clinical Practice guidelines.

Study Design

This phase Ib, multicenter, open-label study enrolled subjects using a standard 3+3 dose escalation design. All patients received letrozole 2.5 mg/d; alpelisib was initiated at 300 mg/d, 25% below the single agent MTD. Both medications were administered daily on a 28-day cycle. In case of adverse events requiring dose adjustments, alpelisib was reduced to 250 mg/d. Intra-patient dose reductions were allowed after the initial 4 weeks of treatment. Patients were treated until disease progression, unacceptable toxicity, or withdrawal of consent.

DLTs were defined as Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 grade ≥3 toxicities. Exceptions were any grade ≥2 toxicity necessitating treatment interruption for more than 21 consecutive days, and non-CTCAE grade 2 hyperglycemia not resolved to grade 0 within 14 consecutive days of initiation of oral anti-diabetic medications (metformin). Grade ≥3 anemia was not considered a DLT unless judged to be a hemolytic process secondary to study drug. Grade ≥3 lymphopenia was not considered a DLT unless clinically significant.

The MTD was defined as the highest dose of alpelisib in combination with letrozole not causing DLT in more than 33% of patients in the first treatment cycle. Twenty or more evaluable patients had to be treated before declaration of the MTD, with ≥6 evaluable patients treated at the MTD for one cycle. Criteria for evaluability were ≥21 days on alpelisib in cycle 1 or early discontinuation due to a DLT. The recommended phase II dose was defined as the highest dose at or below the MTD at which ≥75% of the patients could tolerate therapy for a minimum of 8 weeks without development of grade ≥2 hyperglycemia for more than 14 consecutive days despite initiation of oral anti-diabetic medications; and grade ≥3 rash, grade ≥2 nausea, vomiting or diarrhea, and grade ≥2 rash, all for more than 14 consecutive days of optimal medical treatment.

Safety and Radiographic Assessments

Clinical and laboratory assessments were conducted at baseline and weekly during cycle 1; on days 1 and 15 of cycle 2; and on day 1 of subsequent cycles. Safety assessments included serial electrocardiograms and fasting plasma glucose. Radiographic responses were assessed every two months using RECIST version 1.1. Best response analysis was conducted in any patient that surpassed 8 weeks of treatment, therefore having at least one set of radiographic assessments. Clinical benefit rate was defined as any patient that did not have disease progression on their radiographic assessments for a set time frame (i.e. ≥ 6 or 12 months). Patients that discontinued study treatment due to toxicity prior to their first radiographic assessment were considered non-evaluable.

Mutation Analyses

DNA was extracted using DNEasy or QiaAMP DNA tissue kits from formalin-fixed paraffin embedded (FFPE) archival tumor sections or snap frozen biopsies of metastases, respectively. Tumor cellularity was assessed by an expert breast pathologist (MES); specimens with ≥20% tumor nuclei were considered evaluable. In the few cases with paucicellular samples (<20% tumor cells), multiple sections were macro-dissected to achieve ≥20% tumor cellularity.

DNA was initially subjected to SNaPShot⁽¹⁸⁾ analysis of 18 mutations in PIK3CA, PTEN and AKT1, including the common hot-spot mutations in exons 9 and 20 of PIK3CA, as previously described(19). SNaPShot is based on multiplex PCR, primer extension with fluorescently-tagged dideoxy-nucleotides and capillary electrophoresis; it is a fast, high-throughput, multiplex profiling method based on the Applied Biosystems SNaPshot platform.

Subsequently, Next Generation Sequencing (NGS) was performed using the MSK-IMPACT^™ (Memorial Sloan Kettering - Integrated Mutation Profiling of Actionable Cancer Targets)(20), a hybridization capture-based assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in DNA from FFPE tumor sections. Custom DNA probes targeting exons and selected introns of 341 genes were synthesized using the NimbleGen SeqCap EZ library custom oligo system and biotinylated to allow for sequence enrichment by capture using streptavidin-conjugated beads. Pooled libraries containing captured DNA fragments were subsequently sequenced on the Illumina HiSeq 2500 system (rapid-run mode) as 2 × 100-bp paired-end reads.

FGFR1 fluorescence in situ hybridization (FISH)

Three to four-μm tissue sections were mounted on sialinized slides and hybridized overnight with the ZytoLight SPEC FGFR1/CEN 8 Dual Color Probe (ZytoVision, Bremerhaven, Germany). Briefly, deparaffinization, pretreatment, then the slides were denatured in the presence of 10 μl probe for 6 min at 73°C and hybridized at 37°C overnight in StatSpin (Thermobrite, Abbott Molecular, Inc.). Post-hybridization SSC washes were performed at 72°C and the slides were next stained with DAPI before analysis. Slides were analyzed with Reflected light fluorescent microscope (Olympus BX60) at 100X for hot spots. Representative images of tumor cells were captured using Cytovision software. Thirty tumor cell nuclei from hot spots or random areas, were individually evaluated with the 100X by counting green FGFR1 and orange centromer 8 (CEN8) signals. The average of FGFR1 copy number and the FGFR1/CEN8 was calculated. Cases were considered as FGFR1 positive (‘amplified’) under one of the following conditions:

• a)

  The FGFR1/CEN8 ratio is ≥2.0;

• b)

  The average number of FGFR1 signals per tumor cell nucleus is ≥6;

• c)

  The percentage of tumor cells containing ≥15 FGFR1 signals or large clusters is ≥10%;

• d)

  The percentage of tumor cells containing ≥5 FGFR1 signals is ≥50%, with (a–c) representing a high-level and (d) a low-level amplification(21).

VSports注册入口 - Cell lines

MCF7 and CAMA-1 cells were obtained from ATCC and were cultured in DMEM supplemented with 10% FBS.

Lentivirus transduction

FGFR1 and GFP expressing lentiviruses were generated by co-transfecting 4 μg proviral pLX302-FGFR1 or pLX302-GFP plasmids (Open BioSystems), 3 μg psPAX2 (plasmid encoding gag, pol, rev, and Tat genes) and 1 μg pMDG2 envelope plasmid (Sigma Aldrich) into 293FT cells using Lipofectamine 2000 (Thermo Fisher). 293FT cells were fed with growth media 24 h post-transfection; virus-containing supernatants were harvested 48 and 72 h post-transfection, diluted 1:4 and applied to target cells with 8 μg/mL polybrene (Sigma Aldrich). Target cells were selected with 1 μg/mL puromycin.

Immunodetection

Cells were plated in phenol red-free IMEM + 10% dextran-charcoal-treated FBS and treated for 6 h with drugs or vehicle. Stimulation with FGF2 (Sigma Aldrich) was performed 20 min before the lysis. Lysates were generated by removing media from cells, washing monolayers with cold PBS and lysis with RIPA: 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% Deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM NaF, 1 mM NaVO4, and 1x protease inhibitor mixture (Roche). Lysates were clarified by centrifugation at 15,000 g for 15 min. Protein concentration was determined by BCA assay (Thermo scientific). For immunoblot analysis, equal amounts of protein/lane were subjected to SDS-PAGE, transferred to nitrocellulose membranes and probed with the following antibodies: phospho-Akt (Ser473), total-AKT, phospho-p44/42 MAPK (Erk1/2), total p44/p42 MAPK (Erk1/2), and actin (Cell Signaling Technologies); FGFR1 antibody was obtained from Abcam. Immunoreactive proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

Clonogenic Assays

For longer-term growth assays, CAMA-1 cells were seeded into six-well plates in estrogen free-media (IMEM + 10% dextran-charcoal-treated FBS) plus 5 ng/mL FGF2 and treated with vehicle, alpelisib 2 μM, lucitanib 2 μM (provided by Clovis Oncology), and the combination. Media and inhibitors were replenished every 3 days, and cells were grown for 2 weeks until the untreated wells achieved 80% confluence. Cells were fixed and stained in 20% methanol with 0.5% crystal violet and washed with water. Dried plates were imaged on a flatbed scanner. Afterwards, the crystal violet was solubilized with 20% acid acetic and quantitated by spectrophotometric detection at 490 nm using a plate reader (GloMax®-Multi Detection System, Promega).

"V体育安卓版" Cell Viability

MCF-7 cells were plated in 96-well plates (3,000 cells per well) in a volume of 100 μl of phenol red-free IMEM + 10% dextran-charcoal-treated FBS. Twenty-four h later, DMSO (vehicle) or alpelisib (0.05-4 μM) ± lucitanib 1 μM, all in the presence of 5 ng/mL FGF2 were added to the wells. After 72 h, cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Percentage inhibition was calculated relative to median signal from DMSO-treated wells.

Study Population

Twenty-six patients were enrolled from April 2013 to February 2014 (Table 1). Overall, 85% of the patients had bone metastases and approximately 30% had visceral metastases. All patients not presenting with de novo metastatic disease had previously been exposed to an aromatase inhibitor in the adjuvant setting. In the metastatic setting, 85% had been exposed to at least one line of endocrine therapy, 69% to an aromatase inhibitor, and 31% to chemotherapy. Evaluable tumor samples were obtained in all 26 patients; 74% of these were formalin-fixed, paraffin-embedded (FFPE) blocks from the primary tumor.

"V体育官网入口" Dose Escalation and MTD

Alpelisib dose was started at 300 mg/d. Dose limiting toxicity (DLT) (grade 3 rash) occurred in 2 patients at the 350 mg/d dose. These rashes eventually resolved with medical intervention (high dose anti-histamines, topical and/or oral corticosteroids) and drug interruption. One patient was able to restart at 300 mg/d. The alpelisib MTD was set at 300 mg/d; a total of 20 patients were treated at this dose (escalation and expansion phases). Less than 25% of patients required alpelisib interruption or dose reduction during the first 8 weeks of treatment.

V体育官网 - Safety Findings

Adverse events in this trial were similar to reports from the phase I single agent alpelisib trial(16) and are summarized on Table 2. The most common side effects were gastrointestinal disorders (73%), hyperglycemia (62%), fatigue (54%) and rash (42%); all of these were dose-dependent. Grade 3 adverse events associated with alpelisib were uncommon and generally cumulative (such as hyperglycemia and gastrointestinal disturbances), except for maculopapular rash, which tended to appear within the first 2 weeks of treatment. Of note, rash was less frequent in incidence and severity in patients that had been on anti-histamines (i.e., due to seasonal allergies) prior to trial enrollment. Rashes (of any grade) responded well to high dose anti-histamines (twice daily dosing), obviating the need for corticosteroids in the majority of patients developing this adverse event.

Radiographic Assessments

Twenty-three of 26 patients (88%) were evaluable for best response, 3/26 patients discontinued treatment due to toxicity prior to their first radiographic assessment (Table 3). Five patients (19%) achieved a partial response; four of these patients had ER+/PIK3CA mutated breast cancer. Clinical benefit rate (lack of disease progression ≥6 months) was seen in 9 patients (35%; 95% CI [17%; 56%]), and 8 patients (31%; 95% CI [14%; 52]) continued to have clinical benefit for more than 12 months. Of those 8 patients, 6 had ER+/PIK3CA mutant breast cancer, and all had previous exposure to an aromatase inhibitor in the metastatic setting (Fig. 1). Five patients are still on study for more than 12 months (data cut-off June 2015).

Mutation Analyses

Seventy-five percent (61%) of analyzed tumors were FFPE sections from the original breast cancer diagnosis (median time since original diagnosis 34 months; ranging from 0-99). We initially screened for PIK3CA mutations with SNaPShot⁽¹⁸⁾ (on all 26 tumors’ DNA). We then followed with targeted capture next generation sequencing (NGS; 23 tumors were evaluable for this analysis) in order to identify somatic alterations associated with clinical benefit or lack thereof. In all cases, findings by SNaPShot were confirmed by NGS. NGS analysis showed fewer genomic alterations in tumors from patients with clinical benefit compared to those without clinical benefit (41 vs. 79 genomic alterations; not shown). Most common alterations (>10%) were PI3KCA, TP53, GATA3 and BRCA1/2 mutations, MCL1, FGFR1, CCND1, FGF3/4/19 and CCND1 amplifications, and deletions/truncations in PTEN (Fig. 2A). The majority of PIK3CA mutations were in exons 9 and 20 (E545K, E542K and Q546K, 7 tumors; H1047R alone in 5 tumors). Interestingly, 4/5 (80%) patients with tumors with PIK3CA^H1047R had durable clinical responses, whereas only 2/7 (28%) tumors with mutations in exon 9 exhibited a clinical response in excess of 6 months. One of these tumors had a mutation in the Ras-binding domain (I273V) in addition to E545K. A tumor with a novel deletion in the C2 domain of PIK3CA (P447_L455del) exhibited a solid partial response in liver metastases (Fig. 1).

Seventeen of 23 patients (74%) with NGS data were evaluable for clinical benefit ≥6 months; 3 patients discontinued study treatment due to toxicity prior to their first radiographic assessment; 3 patients discontinued study treatment due to toxicity prior to their second radiographic assessment and were therefore considered non-evaluable for the NGS data/outcome analysis. Seven out of 17 patients (41%) had durable responses to the combination (Fig. 2A). Five of 7 (71%) patients with clinical benefit had a PIK3CA-mutant tumor, in contrast to 5/10 (50%) patients without clinical benefit (Fig. 2B). Two patients with tumors harboring both PIK3CA and a known activating ESR1 mutation (Y537S and D538G, respectively) in their primary tumor exhibited stable disease or partial response. Interestingly, among patients evaluable for response, all those with cancers with FGFR1 and FGFR2 amplification, and KRAS and TP53 mutations progressed on treatment. FGFR1 amplification detected by NGS was confirmed by FISH analysis (Fig. 2C).

FGFR1 overexpression blocks the effect of alpelisib of ER+/PIK3CA mutant cells

To explore whether overexpression of FGFR1 is causal to resistance to alpelisib, we first examined the effect of alpelisib in the ER+ CAMA-1 breast cancer cells, which harbor FGFR1 amplification. FISH analysis of these cells showed a FGFR1/CEN8 ratio of 2.2, consistent with FGFR1 gene amplification (Fig. 3A). Growth of CAMA-1 cells in estrogen-free medium supplemented with FGF2 was completely insensitive to alpelisib, but inhibited by the FGFR1-3 tyrosine kinase inhibitor (TKI), lucitanib (Fig. 3B). Treatment with alpelisib inhibited p-AKT but not p-ERK, whereas lucitanib inhibited p-FRS2 and p-ERK but not p-AKT (Fig. 3C). We then stably transduced ER+/PIK3CA mutant MCF7 cells with a FGFR1 expression vector. Compared to MCF7^GFP control cells, FGFR1 overexpressing cells exhibited slightly higher levels of p-AKT and p-ERK (Fig. 3D). Similar to the results with CAMA-1 cells, treatment with alpelisib inhibited p-AKT but not p-ERK whereas the addition of lucitanib mainly inhibited p-ERK but not p-AKT levels. Finally, MCF7^GFP cells grown as monolayers had an IC₅₀ to alpelisib <1 μM whereas this was at least 10-fold higher in MCF7^FGFR1 cells. Addition of lucitanib resensitized MCF7^FGFR1 cells to the PI3Kα inhibitor (Fig. 3E). Altogether, these data suggest a causal association between FGFR1 overexpression and activation of ERK with relative resistance to alpelisib.