Greipp, Philip R.

Abstract Myeloma is a neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. In these studies, we have demonstrated that CD40 is expressed in human myeloma cells and have used a recently established IL-6-dependent myeloma cell line, ANBL-6, to examine the potential function of CD40 expression in myeloma cells. In addition to its expression on the ANBL-6 cells, we show that CD40 is expressed on freshly isolated myeloma cells from seven of seven patients tested. To address the role of CD40 expression in myeloma cells, we have examined the responsiveness of the ANBL-6 cell line to a CD40-specific mAb, G28-5. This cell line has previously been shown to proliferate only in response to IL-6. Of interest in this study, G28-5 also induced proliferation of the ANBL-6 cells. This proliferation was substantially inhibited by an IL-6-neutralizing mAb. Analysis of ANBL-6 cell culture supernatants by ELISA demonstrated that G28-5-stimulated cells secreted significant levels of IL-6, whereas unstimulated cell culture supernatants contained undetectable levels of IL-6. Furthermore, CV-1/EBNA cells expressing the human CD40 ligand also induced the proliferation of the ANBL-6 cell line, an effect that was inhibited by the anti-IL-6 mAb. Lastly, RNA blot analysis demonstrated an increase in IL-6 message in G28-5-stimulated ANBL-6 cells over unstimulated cells. These results indicate that the primary mechanism of anti-CD40-stimulated proliferation of the ANBL-6 cells is the induction of autocrine IL-6 production. Moreover, these data suggest that the expression of CD40 in malignant plasma cells may play a role in tumor cell expansion, possibly by stimulating the induction of autocrine IL-6 secretion.

8513 Background: Waldenström's macroglobulinemia is a low-grade lymphoplasmacytic lymphoma that is responsive to rituximab. Uniform response criteria define an objective response as a 50% reduction in the IgM level, and a minor response as a 25% reduction in IgM level. No publications in the literature exist that justify the use of the minor response. Clinicians who treat patients that achieve a minor response are left uncertain as to whether the response is adequate and patients should be monitored for progression or whether they should be considered therapy failures and crossover to an alternate chemotherapy regimen in an effort to achieve a deeper response. Methods: 69 patients, 34 previously untreated, and 35 previously treated (but rituximab naive) were included. All patients were treated with a single four-week course of rituximab 375 mg/m2 and were monitored with no further therapy until progression. Results: There were 19 objective and 17 minor responses out of 69 eligible patients (52.2%). Response rate and progression-free survival (26.6 mo) were similar whether patients were previously untreated or previously treated. Previously treated rituximab-naïve and previously untreated patients had, five-year survivals of 48% and 85%, respectively. There was no difference in overall or progression free survival between patients who achieved an objective response when compared to those who achieved a minor response. There was no difference between objective and minor responders by age, time from diagnosis to treatment in the treated group, bone marrow lymphoplasmacytes, hemoglobin level, creatinine, IgM level, or M-spike. The pre-treatment level of IgM did not predict overall survival, progression-free survival, time to progression, or response rate (All p>0.05). This lack of significance was found whether IgM was assessed as a continuous or discrete variable. Conclusions: These results reconfirm rituximab's efficacy as a single-agent for the treatment of Waldenström's macroglobulinemia, and patients who have a 25–50% reduction in their IgM protein derive significant clinical benefits that are durable and appear to not have an impact on overall survival. No significant financial relationships to disclose.

Abstract Introduction: The immunoglobulin free light chain (FLC) is the precursor protein of amyloid in primary systemic amyloidosis (AL). Historically, the ability to monitor the amyloid protein precursor has been crude. There is now a test that reproducibly measures serum levels of FLC. We evaluated the utility of this assay in patients with AL undergoing peripheral blood stem cell transplant (PBSCT). Methods: Between March 1, 1996 and July 31, 2004, 98 patients with AL undergoing PBSCT had serial FLC measurements performed either prospectively or on stored sera. Patients who died before their 100 day evaluation were excluded because response could not be estimated. FLC levels were performed using Freelite™ reagents on a Dade-Behring Nephelometer. In order to pool the data of the monoclonal kappa and lambda patients, the clonal free light chain was considered the involved] immunoglobulin free light chain for the purpose of these analyses and is henceforth referred to as FLC. The prognostic effects of the initial concentration and the extent of reduction of monoclonal FLC on survival were evaluated. Results: Pre-transplant, 94% of patients had an abnormal FLC ratio, with a median involved FLC of 152 mg/L [15.2 mg/dL]. Only 14% of 98 patients had a “measurable” serum M-spike (i.e. ≥ 10 g/L [1 g/dL]) on electrophoresis with a median of 1 g/L [0.1 g/dL]. Ten patients have died of their disease during a median follow-up time of 19.5 months, range 3.3 to 103 months. The 2- year and 4-year estimated survivals are 92% and 76%, respectively. Using the 75th percentile value of pre-PBSCT FLC (382 mg/L [38.2 g/dL]) as a grouping variable, there was a significantly higher risk of death 5.4 (95% confidence interval 1.5–19.3) in the patient quartile with the highest baseline FLC. Baseline FLC correlated with serum cardiac troponin T levels (rho 0.51, p<0.0001), suggesting that high FLC levels are associated with more advanced disease. After PBSCT, the median percent reduction of FLC was 81%. The percent FLC reduction did not predict for survival, but the absolute level of FLC achieved after therapy did. The hazard ratio for death for not achieving a post-transplant FLC of 20 mg/L [2 mg/dL] post-transplant was 5.1 (95% confidence interval 1.1–24.1). Of 91 patients evaluable for complete response by EBMT criteria, 45 had a complete hematological response. Sixty-five percent of the patients achieving a FLC level of less than 20 mg/L [2 mg/dL] also had a complete hematologic response. Free light chain response as defined by a post transplant FLC level 20mg/L [2 mg/dL] or lower was a more powerful predictor of survival than complete hematologic response, as defined by the EBMT multiple myeloma criteria. Post-PBSCT FLC level only weakly predicted for organ response, but median follow-up for surviving patients is relatively short at 19.6 months. Conclusion: FLC measurements both pre- and post-PBSCT are important predictors of patient outcome.

Abstract Purpose: Lenalidomide (CC-5013) is an analogue of thalidomide and a lead clinical compound in a new group of drugs called IMiDS® which have immunomodulatory properties. Lenalidomide has shown promising results in relapsed refractory myeloma. It has a markedly different side-effect profile compared to thalidomide, with significantly fewer non- hematologic adverse events; myelosuppression, especially neutropenia, is the most common toxicity. We report the results of a phase II trial using the combination of lenalidomide plus dexamethasone (Rev/Dex) as initial therapy for newly diagnosed multiple myeloma. Patients and Methods: 34 patients (23 male and 11 female) were enrolled. Lenalidomide was given orally 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone was given orally at a dose of 40 mg daily on days 1–4, 9–12, 17–20 of each cycle. Patients were allowed to go off treatment after 4 cycles of therapy to pursue stem cell transplantation, but treatment beyond four cycles was permitted at the physician’s discretion. For patients continuing therapy beyond 4 months, the dose of dexamethasone was reduced to 40 mg on days 1–4 of each cycle. Objective response was defined as a decrease in serum monoclonal (M) protein by 50% or greater and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours, confirmed by two consecutive determinations at least 4 weeks apart. Sub-classification as very good partial response (VGPR) required in addition to criteria for partial response, >=90% reduction in serum M protein, 24 hour urine M protein less than or equal to 100 mg, and 5% or fewer plasma cells on bone marrow examination. All patients received aspirin (81 mg or 325 mg daily) as prophylaxis against DVT. Results: The median age was 64 years (range, 32–78). All patients were evaluable for response and toxicity. Thirty-one of 34 patients (91%) achieved an objective response to therapy, including 2 patients (6%) achieving a complete response (CR) and 11 patients (32%) achieving very good partial response. Of the 3 patients not achieving an objective response, two met criteria for minor response and one had stable disease. Responses were rapid; the median time to response was 1 month. Adequate stem cells (>3.0 million CD34 cells/kg body weight) were obtained in all patients who proceeded to autologous stem cell transplantation. Forty-seven percent of patients experienced grade 3 or higher non-hematologic toxicity, most commonly fatigue (15%), muscle weakness (6%), anxiety (6%), pneumonitis (6%) and rash (6%). One patient died on study, and this was attributed to infection unrelated to therapy; the patient had stopped all therapy for over a month before the fatal infection occurred. One patient developed a pulmonary embolism, but recovered with therapy; no other patient developed deep vein thrombosis or pulmonary embolism. Conclusion: Rev/Dex is highly active and well tolerated in the treatment of newly diagnosed multiple myeloma with overall responses in over 90% of patients including complete and very good partial responses in 38%. Two large cooperative group trials are currently testing Rev/Dex as initial therapy for multiple myeloma in the United States.

Abstract Background: Although thalidomide induced neuropathy has been described for decades, many features such as incidence, risk factors, severity and character of the neuropathy are not well defined. We retrospectively reviewed the incidence and clinical course of peripheral neuropathy in patients treated with thalidomide or thalidomide/dexamethasone (Thal/Dex) on a Phase II Mayo Clinic trial. Methods: Patients who participated in a Mayo Clinic Phase II trial of thalidomide in myeloma were studied. In this trial, patients with SMM and RMM received thalidomide 200 mg per day escalated as tolerated to a maximum of 800 mg/day. Patients with NMM received thalidomide 200 mg/day plus dexamethasone 40 mg/day orally on days 1–4, 9–12, 17–20 (odd cycles) and 40 mg/day days 1–4 (even cycles) repeated monthly. The incidence and clinical course of neuropathy was assessed by chart review. The study was approved by the Mayo Clinic Institutional Review Board. Results: All 113 patients (70 male, 43 female) treated on trial were studied; 31 patients with smoldering/indolent myeloma (SMM), 32 patients with relapsed/refractory myeloma (RMM) and 50 patients with newly diagnosed myeloma (NMM). Patients were accrued to the parent protocol from April 1999 through March 2002. The median age was 62 years (range, 36–78). The median duration of thalidomide therapy on protocol was 15 months in SMM, 6 months in RMM and 4 months in NMM. Overall, 63 patients (56 %) developed symptoms of peripheral neuropathy. The incidence of neuropathy was 77%, 50%, and 46% in patients with SMM, RMM, and NMM, respectively. Neuropathy worsened during the treatment phase with/without dose reduction or after cessation in 15%, remained stable in 52%, and improved in 27%. Overall grade of neuropathy was grade 1 in most pts; 11% of all patients had grade 2 neuropathy and 2% had grade 3 neuropathy. Predominant symptoms of neuropathy were tingling and numbness involving both upper limb and lower limbs. Small subsets of patients complained of hearing loss 6 patients), and erectile dysfunction (4 patients). Electromyographic studies and nerve conduction studies were performed in 11 patients; the most common finding was a length dependent axonal neuropathy. Conclusion: Peripheral neuropathy is an important and common adverse event with thalidomide therapy that often limits the dose and duration of treatment. Its incidence and severity are likely related to dose and duration of therapy.

Abstract Background: Thalidomide is an active anti-myeloma agent with single agent activity in all stages of the disease. When combined with dexamethasone disease response is seen in nearly two-thirds of patients with newly diagnosed MM. Though initially evaluated for treatment because of its anti-angiogenic properties and the finding of increased bone marrow angiogenesis in MM, it has become clear that thalidomide exerts its anti-myeloma activity through several different mechanisms. While the mechanism of action of dexamethasone has been studied to a great extent in the setting of myeloma, the molecular basis for the activity of this combination is largely unknown. Methods: Myeloma cells from patients with newly diagnosed MM who were enrolled in a phase III trial (ECOG E1A00) comparing thalidomide and dexamethasone combination (T+D) with dexamethasone alone (D) were studied by gene expression profiling. Samples from diagnosis were compared to those at four months following therapy to study the effect of treatment. Four patients who achieved a partial response with T+D were studied and in addition compared to four patients achieving a partial response with D alone. Plasma cells from bone marrow aspirates were separated by magnetic bead selection of CD138 positive cells and studied using Affymetrix HG-U133A chips using standard methodology. The arrays were analyzed using Genespring 7.2 software following GCRMA normalization and genes with differential expression between the two datasets were examined. Differentially expressed genes were further analyzed using Ingenuity Pathways Analysis program Results: 549 genes were two fold or more differentially expressed between the pretreatment and post treatment samples in the T+D group, of which 275 genes were mapped to different networks using the Ingenuity program. Examination of the canonical pathways demonstrated several pathways that were altered as a result of the therapy. The most significant pathways included those related to immune function including antigen presentation (multiple HLA class II antigens), and PRG2 (NK cell activator). Several mediators of B cell signaling including CD45, PKC (α ,β), SHP2, and Erk1/2 were up regulated following treatment. Increase in the expression of several adhesion molecules including β integrins, fibronectin, VCAM1, L selectin, and LFA3 were noted. Up regulation of IL-6 signaling pathway including gp130, JAK2 and IL8 were noted following therapy. Striking increase in the gene expression for CXCR4 and its ligand SDF-1α was noted as well. 129 genes were differentially regulated by dexamethasone alone, of which 67 were mapped to different networks. The pathways influenced by the dexamethasone alone differed significantly from that of the combination. Conclusion: This exploratory analysis offers an interesting insight into the potential mechanisms of action of the T+D combination in MM. The up regulation of immune response related genes are in accordance with some of the mechanisms described for thalidomide. Also, the differences observed between the patients receiving T+D vs. D allows for assessment of the effects of thalidomide. These findings need confirmation in a larger study.

Abstract Background: Lenalidomide has shown efficacy in patients with relapsed myeloma in phase II and III clinical trials, and is currently being investigated as initial therapy for the disease. We report results of a phase III trial comparing lenalidomide plus high-dose dexamethasone (Dex) versus lenalidomide plus low-dose Dex as first line therapy in newly diagnosed multiple myeloma (MM). Methods: Pts with newly diagnosed, untreated, symptomatic MM were eligible. Pts in both arms received lenalidomide 25 mg/day PO on days 1–21 every 28 days. In addition, patients in the high-dose Dex arm (Arm A) received Dex 40 mg on days 1–4, 9–12, and 17–20 PO every 28 days, while pts in the low-dose Dex arm (Arm B) received Dex 40 mg on days 1, 8, 15, and 22 PO every 28 days. The primary endpoint was best response at 4 months on intent to treat basis. At 4 months pts could go off study for stem cell transplant or elect to continue therapy until progression. Response was defined as a decrease in serum and urine monoclonal (M) protein by 50% or higher. If the serum M protein was unmeasurable, a 90% or higher decrease in urine M protein was required. Responses need to be confirmed at least 4 weeks apart. Patients with disease progression or not responding to lenalidomide within 4 months switched to thalidomide with the same dose of dexamethasone they were receiving (Arms C and D, respectively). An independent Data Monitoring Committee approved release of these results. Results: 445 pts were enrolled: 223 randomized to Arm A and 222 to Arm B. Median age was 65 yrs. Serious adverse event data based on expedited reporting (AdEERS) is available on all pts (see table). Common adverse events of Grade 3 or higher were thromboembolism (18.4% in arm A vs 5.4% in Arm B), infection/pneumonia (18.8% vs 9.0%) and hyperglycemia (5.8% vs 1.8%). Incidence of any grade 4 or higher toxicity was 22.0% in Arm A vs 12.6% in Arm B. Response data is being analyzed. Conclusions: Lenalidomide plus two different schedules of Dex was investigated in this phase III trial. Preliminary results suggest that toxicity rates are higher in the high-dose Dex arm. The differences in the response rates between the two arms will dictate future trials and clinical practice. Major Toxicties (AdEERS) Toxicity Arm A (n=223) Arm B (n=222) Cardiac ischemia (Grade >=3) 2.7% 0.5% Hyperglycemia (Grade >=3) 5.8% 1.8% Infection/Pneumonitis (Grade >=3) 18.8% 9.0% Neuropathy (Grade >=3) 0.9% 0.9% Thromboembolism (Grade >=3) 18.4% 5.4% Any non-Hem toxicity (Grade >=3) 53.4% 36.0% Any toxicity (Grade >=4) 22.0% 12.6% Death (Grade 5) 4.5% 1.4%

Abstract Systemic capillary leak syndrome (SCLS) is a vascular disorder characterized by intermittent episodes of hypotension and edema from increased vascular permeability. Patients present with resultant dramatic shifts in plasma volume from intravascular to extravascular spaces. Diagnosis requires observation of severe hemoconcentration and hypoalbuminemia during acute attacks. Typically, laboratory evaluation of hematocrit and albumin is normal when patients are not symptomatic. Concomitantly, and of unknown significance, a monoclonal protein is also frequently discovered. We present the case of a patient whose increased hematocrit suggested polycythemia vera, but whose total clinical picture of hypotension, hemoconcentration and hypoalbuminemia confirmed the diagnosis of SCLS. A 54-year old gentleman was referred for evaluation of polycythemia vera that was diagnosed after a series of dramatic hospitalizations for near-syncope. The patient had no chronic illnesses, negative family history for hematologic abnormalities, and was otherwise in his usual state of good health when a complaint of sudden lightheadedness and pre-syncope led to an emergency room evaluation. There he was found to be severely hypotensive and was resuscitated with intravenous fluids. Initial laboratory evaluation showed a hematocrit of 60%. Further workup revealed an increased red cell mass. For the presumptive diagnosis of polycythemia vera, the patient was phlebotomized once and, interestingly, did not require any further phlebotomy for almost three years. During this interim, he remained relatively asymptomatic with only occasional bouts of lightheadedness that would abort with increased fluid hydration. Phlebotomy was again used when the patient presented with near-syncope, hypotension, and vomiting accompanied by significant extremity swelling. The hematocrit was found to be greater than 80%. The patient recovered with aggressive fluid resuscitation and was discharged on a low-dose aspirin with the continued empiric diagnosis of polycythemia vera. During that year, the patient recovered from four other similar episodes that resolved after fluid resuscitation and where phlebotomy was used. The most severe episode involved a protracted ICU stay for severe hypotension complicated by severe extremity edema and resultant compartment syndrome and ischemic muscle necrosis. With the concern of adrenocortical dysfunction leading to these hypotensive episodes, the patient was started on low dose daily steroids. While on this, the patient reported less frequency and severity of his almost-daily symptoms of lightheadedness and edema. In fact, attempts at tapering the steroid dose down resulted in a return of the minor symptoms and with continued steroids, no further hospitalizations were needed. During our evaluation, we confirmed a normal hematocrit and albumin while asymptomatic and the presence of a monoclonal protein. This case represents a common clinical history of patients who are ultimately diagnosed with SCLS. Often, the significance of the observed hypotension in concert with laboratory evidence of hemocentration and hypoalbuminemia is not initially appreciated. Although current attack prophylaxis regimens for SCLS use terbutaline and theophylline, steroids had historically shown occasional benefit in some patients. SCLS still carries a significant mortality risk and prompt recognition of this syndrome and initiation of an appropriate prophylaxis regimen is crucial.

Abstract Fluorescence in situ hybridization (FISH) is an effective alternative for the analysis of cytogenetic abnormalities of low proliferative entities such as Waldenström macroglobulinemia (WM) but, to date, no studies have been carried out to explore the potential impact of molecular cytogenetics on disease evolution. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. In this study we have analyzed the incidence of this aberration (using both conventional cytogenetics and FISH analysis) in 102 WM patients, and we have correlated it with disease characteristics. Deletion of 6q21 was detected in 6% of patients by conventional cytogenetics, while FISH studies increased this incidence up to 39%. Moreover, in those patients analyzed by cIgM-FISH, the incidence of 6q- was higher (54%). Interestingly, 6q- patients displayed features associated with adverse prognosis (higher levels of ESR, CRP and beta-2 microglobulin), and a greater tendency to display anemia and hypoalbuminemia. Other differentiating variables without reaching statistical significance were a tendency of displaying a higher bone marrow lymphoplasmacytoid infiltration and the amount of monoclonal component in 6q- patients (table). In addition, patients with 6q- displayed shorter survival from time of diagnose (log-rank; p=0.09). In summary, our results show that 6q deletion in WM patients is currently the most frequently found cytogenetic abnormality. Patients with this deletion also shows a trend towards an aggressive disease and poorer outcome in WM. Further studies should focus on possible tumor suppressor genes in the minimal area of deletion at 6q and to determine their role in disease progression. table del6q present del6q absent p-value n 40 (39%) 62 (61%) β2M ≥4mg/dL 48% 16% 0.005 CRP ≥3.5 mg/dL 47% 11% 0.006 Albumin <4mg/dL 86% 55% 0.006 ESR ≥100 mm/h 73% 44% 0.02 Hb <11 g/dL 63% 40% 0.02 serum IgM >20 g/L 86% 73% 0.10 BM infiltration >40% 44% 31% 0.20

Abstract Patients with multiple myeloma show significant heterogeneity with respect to progression, therapeutic response and toxicities. Part of the Bank On A Cure project is to examine potential genetic polymorphisms that may be associated with common toxicities monitored in clinical trials. The SWOG/ECOG intergroup trial S9321 therapy includes VAD induction followed by randomization to high dose melphalan with autologous transplant support. Paticularly important toxicities with VAD include infection, thrombosis and hyperglycemia; and with melphalan toxicities in GI function are noted. Toxicities and genotypes were assessable during VAD induction on 569 patients; and during high dose melphalan on 161 patients. Toxicities examined included an array of symptoms: cardiovascular, flu-like, GI, neurologic, infection, lung, metabolic, and pain. Grade 3 toxicities found in at least 20 patients were compared to SNP genotypes. For VAD induced toxicities,TNF-alpha SNP genotypes showed association with fatigue, dyspnea and thrombosis; CYP3A4 SNP genotypes showed association with fatigue; DNA repair SNP genotypes in XRCC1 showed associations with infection, hyperglycemia and dyspnea; LT-alpha SNP genotypes showed association with dyspnea; and IL-6 and IL-10 SNP genotypes were marginally associated with neuropathy. After high dose melphalan, diarrhea was associated with SNP genotypes in TGF-beta, CYP3A4 and ERCC2. Interestingly, IL-6 polymorphisms were significantly associated with infection. The p values on the above associations ranged from .05 to .004. In addition, we noted ethnic distributions of some SNP genotypes were significantly different; and significant ethnic disparities in therapy induced hyperglycemia, fatigue, dyspnea and thrombosis were noted (p values range from .04 to .009). Of particular note is that the high producer IL-6 genotype is present in over 90% African Americans, compared to 35% Caucasians. Details of each association will be presented. Not all associations can easily be explained by known biologic function of the genotype, and it is important to qualify these results, as the likelihood of seeing some genotype associations by chance is quite high given the number of comparisons made. Further, we are continuing to examine additional prognostic factors that may, together with genotypes, provide collective predictors of toxicities. We consider these associations as targets for further study through the increasing sample acquisition in the Bank On A Cure project.