Phosphorylation Levels of Extracellular-Signal Regulated Kinase (ERK) and AKT in Circulating Lymphocytes Predict Response to Targeted Therapy with Kinase Inhibitors in Refractory/Relapsed Hodgkin Lymphoma Patients,

Identification of predictive biomarkers of response to molecularly targeted therapy represents a challenging issue. Therefore, we conducted a pharmacodynamic study evaluating phosphorylation levels of ERK (pERK) and AKT (pAKT) in circulating lymphocytes from patients with refractory/relapsed Hodgkin lymphoma (HL) enrolled in two consecutive phase II trials evaluating activity and safety of the multikinase inhibitor Sorafenib (Nexavar^®, Bayer) used as single agent, or in combination with the AKT inhibitor Perifosine (Æterna Zentaris GmBH, Germany).

Between July 2008 and May 2011, 25 patients (15 males and 10 females; median age 36 years, range, 18–73 years) with relapsed (n = 3) or refractory (n = 22) HL were analyzed. In one trial, patients received Sorafenib alone (400 mg BID, per os), and in the second trial Perifosine (50 mg BID, per os) for 4 weeks followed by combination therapy of Perifosine plus Sorafenib (400 mg BID, per os) until disease progression or clinically significant toxicity. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. Peripheral blood lymphocytes (PBL) collected prior to therapy initiation and monthly thereafter were analyzed for pERK and pAKT expression by flow cytometry. Unstimulated or phorbol myristate acetate (PMA)-stimulated PBL were fixed with paraformaldehyde, permeabilized with methanol and stained with Alexa Fluor-conjugated Anti-ERK1/2 (pT202/pY204) and anti-Akt (pS473) monoclonal antibodies (MoAbs) or the appropriate isotype controls (all from Becton-Dickinson). PBL from normal donors were also included as internal control. Results were expressed as percentage of cells stained with the relevant MoAb in PMA-stimulated cultures after subtraction of staining detected in unstimulated PBL. The study was approved by the Institutional Ethical Committee.

Eight out of 25 patients (32%) achieved objective responses, including complete response (CR, n = 1) and partial response (PR, n = 7), whereas 9/25 patients achieved stable disease (SD), and 8/25 patients experienced PD. Median overall survival (OS), progression-free survival (PFS) and duration of response (DOR) were 16, 5 and 4 months, respectively. Data of pERK and pAKT were available for 23 and 20 patients, respectively. At baseline, the median pERK level was 43% (range, 6 – 82). Baseline pERK levels were significantly higher in responsive patients, i.e., those achieving CR, PR and SD (n = 14) as compared to unresponsive patients (n = 9), i.e., those experiencing PD, (55 ± 18% vs 22 ± 9%, P =.0001). The median baseline value of pERK efficiently discriminated responsive and unresponsive patients. In fact, 12/12 patients with baseline pERK levels ≥43% achieved CR, PR or SD, whereas 9/11 patients (82%) with baseline pERK <43% experienced PD (P <.0001). Moreover, median PFS was 9 months for patients with baseline pERK ≥43%, and 3 months for patients with baseline pERK <43% (P <.0007). After 2 months of therapy, pERK levels were significantly reduced in responsive patients (P =.0001), whereas they were unchanged in unresponsive patients. Similarly, significantly higher baseline levels of pAKT were observed in responsive (n = 12) as compared to unresponsive (n = 8) patients (42 ± 14% vs 15 ± 6%, P ≤.0001), with a significant reduction of pAKT levels being observed after 2 months of therapy in responsive patients only. The median baseline value of pAKT discriminated responsive and unresponsive patients (P =.02). In fact, patients with pAKT levels ≥23% (n = 10) had significantly longer PFS than patients with baseline pAKT levels <23% (7 vs 3 months, P ≤.008). Assuming the median baseline levels of pERK (43%) and pAKT (23%) as cut-off, the positive predictive values were 100% for pERK and 90% for pAKT, and the negative predictive values were 82% for pERK and 70% for pAKT. Sensitivity of pERK ≥43% and pAKT ≥23% were 86% and 75%, respectively, with a specificity of 100% and 87%, respectively.

Clinical data from phase II studies using Perifosine and/or Sorafenib in patients with relapsed/refractory HL patients suggest a correlation of baseline pERK and pAKT levels with objective responses and time to tumor progression. Whether these parameters will prove to be useful predictive biomarkers of response in HL patients receiving kinase inhibitors will need to be validated in prospective studies.

Off Label Use: Sorafenib in Hodgkin Lymphoma Patients.