The AKT/mTOR/P70S6K/4EB-P1 Signaling Pathway Is Activated in a Subset of Multiple Myeloma Patients and Correlates with High Serum Levels of Beta2-Microglobulin

Introduction: The mammalian target of rapamycin (mTOR) is a serine/threonine-specific protein kinase, downstream of the phoshatidylinositol 3-kinase (P13-K/AKT) pathway. Constitutive activation of the mTOR related upstream and downstream effectors including P13-K, AKT, P70S6K and 4E-BP1 have been found in numerous malignancies. Previous studies demonstrated that rapamycin has preclinical potential as therapy for multiple myeloma (MM), especially when associated with other drugs.

Methods. We performed immunohistochemical analysis with p-AKT (Ser 473), p-mTOR (Ser2448), p-P70S6K (Thr389) and p-4E-BP1 (Thr37,Trh46) on bone marrow sections of 73 symptomatic MM patients. Mielomatous plasmacells were identified and counted by mouse monoclonal CD138 nd all cases were analyzed using a semiquantitative histologic score (HSCORE) method. Specifically, immunostaining intensity of each case was semiquantitatively scored as follow:

• 0, no staining;

• 1,weak staining;

• 2, moderate staining; and

• 3, strong staining.

For each case, a value designed HSCORE was obtained multiplying each intensity with the corresponding percentage of positive cells [HSCORE =∑(1XPC), where 1 and PC represent intensity and percentage of cells, respectively]. Specimen with an HSCORE of ≥30 were classified as p-AKT, p-mTor, p-P706SK and p-4E-BP1 positive. Wilcoxon test was used to compare mTOR expression with clinical data of all patients (including age, presence of bone lesions, isotype, Beta2-microglobulin, haemoglobin, creatinine and albumin serum levels). Common cytogenetic abnormalities (t(11;14), t(4;14), del 13q14 and del p53) were also detected in 61 of 73 (83.5%) patients by FISH analysis on CD138 purified plasma cells.

Results. Fouty-four (60.2%) and 46 of 73 (63%) patients stained positive for p –AKT and p-mTOR with a cytoplasmic staining pattern, respectively. P-mTOR immunoreactivity was strongly, moderately and weakly positive in 23.9 %, 34.8 % and 41.3 % of the 46 positive samples, respectively. P-P70S6K and p-4E-BP1 was detected in 53 (72.6%) and 40 (54.8%) patients with a predominantly nuclear staining pattern. The intensity of positivity was distributed as follows: p-P70S6K strongly, moderately and weakly positive, 54.7 (%), 26.4 (%) and 18.9 (%),respectively; p-4E-BP1 strongly, moderately and weakly positive, 62.5 (%), 17.5 (%) and 20 (%), respectively; Of the 46 myelomas stained positive for p-mTOR, 35 expressed p-AKT, 33 expressed p-4E-BP1 and 40 demonstrated p-P70S6K positivity. P-mTOR expression significantly correlated with p-AKT (p=0.003), p-P70S6K (p<0.001) and p-4E-BP1 (p< 0.001) staining consistent with the hypothesis that the AKT/mTOR/P70S6K/4E-BP1 pathway is activated in a subset of MM cases. A significant statistical correlation was found between mTOR expression and serum levels of Beta2-microglobulin ≥3 g/dl (P=0.039). No clearly correlation was found between mTOR pathway activation and the molecular cytogenetic anomalies.

Conclusion: We identify a subgroup of MM patients with mTOR pathway activation and poor prognosis. Phosphoprotein staining of the mTOR pathway should be studied further as a means to select patients to receive mTOR inhibitors in combination of chemotherapy. These data also strongly suggest that the AKT/mTOR/P70S6K/4E-BP1 pathway activation may constitute a new biologic parameter regardless of molecular cytogenetic.