Introduction: Lenalidomide (LD) is an oral thalidomide analog with both immunomodulatory and anti-angiogenic properties. It is approved for use in myelodysplastic syndromes (MDS), specifically low-risk and intermediate 1 with 5Q-. LD has also been approved for relapsed multiple myeloma (MM). A major complication associated with LD includes venous thromboembolism (VTE), especially in combination with steroid and erythropoetin (EPO). VTE occurs in 2% of MDS, but range from 6.6% as monotherapy in MM to 8.5%–13.5% when combined with dexamethasone. Zonder reports a preliminary incidence of 75% (9/12 patients) in the SWOG 0302 trial of LD and dexamethasone in previously untreated myeloma. With the addition of ASA in these patients the incidence dropped to 19%. Patients with polycythemia rubra vera (PRV) have a higher incidence of VTE (5.1%> age 70). JAK-2 mutation (tyrosine kinase mutation) occurs as a sporadic event in 75% of patients with PRV and is associated with an older age group, inferior survival, myelofibrosis, acute leukemia and thromboembolic events. Recent studies with LD in this group of PRV and spent-phase myelofibrosis have shown encouraging results. LD has an activity of 46% in myelofibosis by decreasing transfusions and splenomegaly. It would seem to have activity in spent-phase PRV. We report a seminal event of splenic infarction in a patient with PRV and myelofibrosis treated with LD. Case Presentation: A 73 y/o male was diagnosed with PRV 16 yrs earlier. He underwent phlebotomy and received ASA. With a hyperproliferative phase he received hydrea as he was intolerant of IFN-A and anagrelide. Three months earlier, he developed fever, sweats, splenomegaly, and became transfusion dependent. His marrow confirmed myelofibrosis with dysplastic features. LD was begun at 25 mg daily. Three weeks later he developed pneumonia with bronchospasm and required hospitalization for antibiotics and steroids. He improved but returned 4 days later with left upper quadrant pain with CT findings consistent with multiple splenic infarcts. Prednisone was stopped and enoxaparin resulted in marked improvement. Currently, he has marked diminution in the size of his spleen from 13 cm to 4 cm. Currently, he is transfusion independent with marked clinical improvement. Discussion: This report points several dynamic issues within the field of myeloproliferative syndromes. The JAK-2 mutation offers fertile areas of research within MPD disorders as a causative or sporadic mutation and as an associated with several adverse events including VTE and myelofibrosis. We report here a unique occurrence of VTE in the form of multiple splenic infarctions within the context of treatment of the spent-phase of PVR with JAK-2 mutation receiving LD. VTE events associated with LD are associated with DVT or pulmonary embolus and have not been associated with splenic infarction. Splenic infarction has also been reported in myelofibrosis. As further studies confirm efficacy with myelofibrosis it should be suggested that prophylaxis with warfarin or enoxaparin, especially in JAK-2 mutation patients, be considered. Physicians should be encouraged to report their experience in patients with MPD and lenalidomide.

INTRODUCTION: Primary amyloidosis is a plasma cell dyscrasia related to multiple myeloma. The treatment for primary amyloidosis is very similar to the treatment used for multiple myeloma. A new agent, bortezomib, has shown promising results for the treatment of multiple myeloma, but has not yet been used for the treatment of primary amyloidosis. We report a case of a patient with primary amyloidosis who was treated with bortezomib as a 2nd line agent and had significant clinical and radiological regression of disease. CASE PRESENTATION: A 56 yr old male presented with complaints of anorexia, weight loss, shortness of breath, and parathesias, and RUQ pain. Physical examination and subsequent radiologic studies showed an enlarged liver mass. Laboratory data was significant for elevated alkaline phosphatase and an IgG lambda monoclonal gammapathy in both the urine and serum. A liver biopsy revealed diffuse infiltration of hepatic parenchyma by amyloid deposits. A bone marrow examination revealed 7% plasma cells. His urine immunoelectrophoresis confirmed the presence Bence Jones proteins and a monoclonal IgG lamda spike. Additional work up revealed that he had both renal and cardiac involvement. The patient was treated with alkeran 12 mg days 1–4, thalidomide 200 mg, and dexamethasone 20 mg days 1–4, 9–12, and 17–20, with a good response initially. Seven months into treatment the patient developed a gastrointestinal bleed requiring colonoscopy. Endoscopy and biopsy findings revealed amylodosis involvement of the colon. The patient was re-treated with the same regimen, this time with a partial response, but the medications were discontinued secondary to the worsening of paresthesias and postural hypotension. 2 ½ years after his initial diagnosis, bortezomib was initiated. An excellent clinical and radiological response was seen, with a reduction in the hepatic size from 20 cm to 10 cm, improved cardiac and renal function, and disappearance of the monoclonal protein from his urine. DISCUSSION: In the past few years the advances in the treatment of primary amyloidosis has suggested that intensive chemotherapy may produce disease remission and dramatic clinical improvement in selected patients. Unfortunately, the rarity and rapid progression of this disease often delays diagnoses until multiorgan involvement limits the ability to treat. Due to the similarities in treatment between amyloidosis and myeloma, data was extrapolated from studies conducted in myeloma, and subsequently applied in this patient. A randomized phase III multicenter trial compared bortezomib with high dose dexamethasone in 669 patients with relapsed/refractory myeloma (APEX™ trial). The overall response rate (38 versus 18 percent) and complete (6 versus 0.6 percent) response rates were significantly higher in patients receiving bortezomib. Patients receiving bortezomib demonstrated a median time to progression of 6.2 months versus 3.5 months for those receiving dexamethasone. Estimated one-year survival was significantly longer for bortezomib-treated patients (80 versus 66 percent). The positive results from this trial, suggested that bortezomib might also have a beneficial role in the treatment of primary amyloidosis. This case shows a documented regression of this patient’s amyloidosis due to bortezomib treatment, further studies of bortezomib in primary amyloidosis need to be performed to validate this possibility.

Bortezomib (PS341, Velcade) is a selective, reversible proteosome inhibitor. It is a unique inhibitor of the ubiquitin-proteosome pathway leading to arrest in tumor growth, induction of apoptosis, inhibition of tumor metastasis and angiogenesis. It plays an important role in the activation of NF Kappa B, which is responsible for transcription of inhibitors in apoptosis. It is a novel therapeutic agent that has demonstrated in vitro and in vivo activity in mantle cell lymphoma (MCL). In one phase II study, O’Conner et al reported that bortezomib demonstrated a response rate of 50% in refractory cases of mantle cell lymphoma, where as in small lymphocytic lymphoma there was a 0% response rate. Why certain types of non-Hodgkins lymphoma (NHL) are sensitive to this agent and others resistant is currently not well understood. It is believed that disregulation has been shown to play a role in the development of drug resistance in NHL Our objective was to determine the efficacy of bortezomib in vitro using three subtypes of NHL. Three lymphocytic cell lines were used: Granta-519 (mantle cell lymphoma), Toledo (diffuse large cell lymphoma) and EHEB (chronic B cell leukemia). The cytotoxic effects were determined by the colorimetric MTT assay after 24 hours of treatment with bortezomib (1 – 20 nM). Apoptosis was assessed by measuring Caspase-3 activity after 6 and 16 hours of treatment with bortezomib (4 or 20 nM). Bortezomib demonstrated a dose-dependent cytotoxic effect for all three cell lines, with an IC50 of 3.5 nM, 4.1 nM, and 18.5 nM for Toledo, Granta-519 and EHEB, respectively. Apoptotic studies showed that bortezomib (20 nM) induced a 1.4 to 5.6-fold increase (after 6 hrs) and 5.8 to 16.6-fold increase (after 16 hrs) in apoptosis. The EHEB cell line was most resistant, showing no increase in Caspase-3 activity at the lower dose (4 nM) at either time-point. Granta-519 was most sensitive showing a 5.2-fold increase in apoptosis with the lower dose after 16 hours of treatment. This in vitro study demonstrated a dose and time dependent response of bortezomib in both the mantle cell lymphoma (Granta-519) and the diffuse large cell lymphoma (Toledo) cell lines. The resistance to bortezomib in the chronic B cell leukemia (EHEB) cell line was confirmed in this study as evidenced by the lack of Caspase-3 activity at the 4 nM dose at both 6 and 16 hours. Our future studies will attempt to unmask the reason for this resistance. Perhaps manipulation of the apoptotic pathway through the use of combinations of various biologic response modifiers may be an attractive option in overcoming resistance in certain subtypes of NHL.

Background: Bortezomib is a selective, reversible inhibitor of the ubiquitinproteasome pathway whose action leads to tumor growth arrest and induction of apoptosis. Bortezomib has demonstrated clinical activity in Non-Hodgkin lymphoma (NHL) and is approved in the treatment of mantle cell lymphoma (MCL). The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a key role in the PI3K/AKT pathway that regulates tumor cell growth and proliferation. Temsirolimus is an mTOR inhibitor that has shown activity in preclinical and clinical studies in various NHL subtypes. The objective of this study was to determine the growth inhibitory and apoptotic effects of the novel combination of bortezomib and temsirolimus in selected NHL cell lines including: Granta-519 (MCL), Toledo (large B-cell lymphoma), and EHEB (chronic B-cell leukemia). Materials and Methods: Cell viability was determined by the colorimetric MTS assay after 24 and 48 hours of treatment with bortezomib (1 – 10 nM) and temsirolimus (0.1 – 5 μM). Drug interactions were examined by the combinationindex (CI) isobologram method of median dose-effect analysis. Apoptosis was assessed by measuring cytochrome c and caspase-3 activity after 24 hours of treatment. In addition, the expression of several key factors involved in pro-apoptotic signaling (Bax and FasL) were evaluated by western blot analysis. Results: The MTS assays revealed a significant time and dose-dependent growth inhibition with either agent alone in all three NHL subtypes. A synergistic interaction was demonstrated with bortezomib and temsirolimus in the EHEB and Granta-519 cell lines, while an additive effect was seen at lower drug doses in the Toledo cell line. The LD50 ranged from 2.7 – 9.2 nM for bortezomib alone and 0.3 – 10.6 μM for temsirolimus alone. These levels were reduced to 0.4 – 4.5 nM and 0.04 – 2.25 μM for bortezomib and temsirolimus, respectively, when these drugs were used in combination. Growth inhibition in these cell lines was due in large part to the induction of the intrinsic apoptotic cascade. A 4 to 10-fold (bortezomib) and 2 to 8-fold (temsirolimus) increase in caspase-3 activity was seen, which was significantly increased 8 to 24-fold (p < 0.001) using the drug combination. In addition, bortezomib was shown to induce mitochondrial cytochrome c release in the 3 cell lines, while temsirolimus initiated release in the EHEB and Granta-519 cells. Increased expression of Bax, a pro-apoptotic member of the BCL-2 family of proteins, was observed predominantly in the MCL subtype (Granta-519) following treatment with either agent. This expression was further enhanced using both drugs. Additionally, an increase in FasL expression in all cell lines with the drug combination suggested an activation of the extrinsic apoptotic pathway. Conclusion: Targeting both the ubiquitin-proteasome and mTOR pathways, which are critical for tumor cell growth and survival, may be a promising new therapeutic strategy in NHL. This in vitro study demonstrates a significant increase in growth inhibition and apoptosis with the novel combination of bortezomib and temsirolimus, when compared to either drug alone, in selected NHL subtypes. These results provide a rationale for the use of this drug combination in NHL clinical trials.