Lacy, Martha Q.

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 Introduction: The association of amyloidosis with immunoglobulin light chain disorders is well recognized. It is less commonly appreciated that amyloidosis can be associated with IgM monoclonal gammopathies. One of the available therapies for treating amyloidosis is stem cell transplantation. It is unknown whether the clinical outcomes in patients with IgM amyloidosis differs from those patients with light chain amyloidosis. Patients: Among 242 amyloidosis patients receiving stem cell transplants 10 (4%) had an IgM monoclonal protein. Eight were Mλ. One was biclonal Mλ Mκ and one was biclonal Mκ Gλ. The age range was 56 to 71, median 64 years. Seven were men. The monoclonal protein ranged in size from 0.1 to 2.3 g/dL (median 1.2 g/dL). Patients were transplanted from 2 to 8 months (median 4 months) following histologic diagnosis of amyloid. The median time from the recognition of the IgM monoclonal protein to the diagnosis of amyloid was 1.3 months, but 2 patients, both of whom had overt Waldenström’s macroglobulinemia, were diagnosed 45 and 115 months respectively before amyloidosis developed. Of the cohort of 10, 3 fulfilled the criteria for symptomatic Waldenström’s macroglobulinemia. Results: Amyloid syndromes were recognized in the kidney, heart, peripheral nerve and liver in 8, 3, 3 and 1 patient, respectively. Of the 3 peripheral neuropathy patients one also had autonomic failure. Seven of the 8 renal amyloid patients were confirmed with a renal biopsy. Their urinary protein loss ranged from 0.1 to 9.5 g/24h with a median of 4.7 g/24h. Six of the 10 patients had single organ involvement, 3 had 2 organ involvement and 1 had 3 organ involvement. All 3 Waldenström’s patients had prior exposure to cytotoxic chemotherapy. Amyloid deposits were seen in the bone marrow, fat and lymph node in 6, 5 and 1 patient, respectively. Patients underwent from 1 to 7 (median 3) apheresis with a median CD34 yield of 4.5 x 106/kg. Conditioning was Melphalan at 140 and 200 mg/m2 in 2 and 8 patients respectively. Neutrophil engraftment 500/μL occurred between day 11 and 30 (median 14) and for platelets 20,000/μL between day 11 and 406 (median day 16). The median number of hospitalization days ranged from 0 to 32 (median 12). Nine of the 10 patients fulfilled criteria for a hematologic response and to date 3 of these 9 have also achieved an organ response, including normalization of urinary protein loss in 2 nephrotic patients and a greater than 50% reduction in urinary protein loss in the third. None of the patients with amyloid neuropathy responded. One of the patients with cardiac amyloid showed echocardiographic evidence of improvement. At this time 8 survive and 2 died; 1 a treatment-related death (day +40) and 1 of relapse post transplant with progressive amyloid at 45 months. The median follow-up of surviving patients is 29 months. Conclusion: The outcomes in patients with IgM amyloidosis following transplantation do not appear to be different from those with immunoglobulin light chain amyloidosis. There is no suggestion that the response rates are lower or that alternate conditioning schemes such as BEAM should be considered.

Abstract INTRODUCTION Stem cell transplant has been increasingly used in the initial therapy of newly diagnosed systemic AL. Introduction of the free light chain assay has made it easier to quantify responses in patients with AL. It is unclear whether the goal of therapy should be a hematologic response or, as in multiple myeloma, a complete response. This paper addresses the impact of the degree of response on outcome. PATIENTS AND METHODS 282 consecutively transplanted patients are included in this analysis. None of the patients were excluded. There were 169 men (59%). 28% of the cohort have died. 69% had renal involvement, 51% cardiac involvement, 11% peripheral nerve involvement, 16% hepatic involvement. The median percentage of plasma cells at the time of transplant was 7. Echocardiographic analysis revealed a median septal thickness of 12 and a median ejection fraction of 65%. Patients ranged in age from 31 to 71 with a median of 57 years and were transplanted at a median of 4.2 months following histologic diagnosis of amyloidosis. 27% of patients received reduced intensity conditioning usually due to moderate cardiomyopathy or renal insufficiency. 201 patients achieved a hematologic response and 93 of these achieved a complete response. The criteria for response were reported in accord with the consensus definition for response established at The Xth International Symposium on Amyloid and Amyloidosis. A complete response required immunofixation negativity of serum and urine with a normal free light chain ratio. A hematologic objective response required a 50% reduction of serum and urine monoclonal protein with a 50% reduction in the free light chain value. Figure 1 shows the Kaplan Meier curves demonstrating the survival of all patients based on whether they achieved a complete response, partial response, or failed to achieve response. There were 93 CR, 108 PR, and 81 nonresponders. The differences in all three curves are significant at P<.001. Figure 2 demonstrates the same cohort of patients using a six month landmark analysis to exclude the effect of treatment related mortality on outcome. There were 86 CR, 91 PR, and 36 NR for a total of 213 patients. There was a difference in response rate based on conditioning intensity (p<0.01) and age (p=0.046). The median survival was only reached in the NR at 40.1 months and, in the landmark analysis the differences between all three survival curves were statistically significant. CONCLUSION Patients who achieve a hematologic complete response following sct for AL do better than other patients. Patients who achieve an objective response have a superior outcome than patients who fail to achieve a hematologic response, in a landmark analysis at six months. It is unclear if consolidation chemotherapy should be offered to partial responders in an attempt to convert them into CR’s. Figure Figure Figure Figure

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 AMD3100, a CXCR4 receptor antagonist, has been studied as a stem cell mobilization agent for the purpose of autologous stem cell transplantation (ASCT) in hematologic malignancies. Lymphocyte subset analysis of peripheral blood in patients treated with AMD3100 has been studied in healthy volunteers. To date, no reports exist describing the lymphocyte subsets of the autograft in patients undergoing AMD3100 stem cell mobilization for the purposes of ASCT in non-Hodgkin lymphoma (NHL). Considering our prior work demonstrating the significant impact of autograft lymphocyte content on clinical outcomes of patients undergoing ASCT for NHL we set out to profile autograft lymphocyte subsets of patients undergoing mobilization with AMD3100. Using flow cytometry, we analyzed aliquots of apheresis products in 7 patients with NHL undergoing AHSCT who received AMD3100 in addition to G-CSF as a part of their mobilization regimen. These results were compared to 29 patients with NHL who had undergone stem cell mobilization with G-CSF alone. There were no significant differences between these two groups of patients in terms of sex, age, performance status, histology, LDH, and number of pretransplant chemotherapeutic regimens. CD34+ cells collected at apheresis did not differ significantly between the groups. However, compared with G-CSF alone, patients that received AMD3100 had an approximate 5-fold increase in CD4+ cells (0.62 x 109 cells/kg vs. 0.12 x 109 cells/kg, p = 0.0004), a 3.5-fold increase in the absolute number of autograft CD3+ cells (1.21 x 109 cells/kg vs. 0.33 x 109 cells/kg, p = 0.0017), and a 2.5-fold increase in CD8+ cells (0.48 x 109 cells/kg vs 0.19 x 109 cells/kg, p = 0.215). A significant increase was also noted in CD4+25+ cell compartment (0.17 x 109 cells/kg vs. 0.006 x 109 cells/kg, p = 0.0001). No significant difference was noted in the absolute number of autograft CD16+56+ NK cells. Finally, an increase in the autograft total absolute lymphocyte count (41.6 x 109 cells/kg vs. 2.88 x 109 cells/kg, p < 0.001) as well as peripheral blood absolute lymphocyte count at day 15 after AHSCT was observed in those patients who had received AMD3100 (0.79 x 109 cells/kg vs. 0.58 x 109 cells/kg, p = 0.04). The increased lymphocyte content of the autograft (total and subset) would suggest the potential of positive impact on clinical outcomes in patients mobilized with AMD3100. Indeed, none of the patients who received AMD3100 had relapsed disease at one year post-transplant (although two of these patients are currently 9 months and 11 months post-transplant respectively), whereas 10 of the 29 control patients had relapsed disease at one year. Further studies are necessary to confirm these observations and ascertain their clinical significance.

Abstract Background: While the therapeutic armamentarium for plasma cell disorders have expanded recently with introduction of thalidomide, lenalidomide and bortezomib, these diseases are still incurable and more agents are needed. We have previously demonstrated expression of CD 52 on plasma cells in MM, MGUS and AL amyloidosis. Based on this data we conducted a Phase II Study of Campath (Alemtuzumab; Monoclonal Anti-CD52 Antibody) in Primary Systemic Amyloidosis and Relapsed or Refractory Multiple Myeloma. Methods: The study was an IRB approved, Phase II protocol, with a two stage design and interim analysis at 14 patients, to assess the efficacy and toxicity of Alemtuzumab. The regimen involved a dose escalation during week one (Day I 3 mg SQ; Day II 10 mg SQ; Day III 30 mg SQ; then 30 mg SQ three times a week for a planned total of 17 additional weeks). All patients received concurrent Co-Trimoxazole, Fluconazole and Valacyclovir prophylaxis. The planned accrual was 32 patients. Results: A total of 14 patients were accrued, 8 patients with MM and 6 patients with AL. There were 8 females and 6 males in the study. The median age was 62 years (range: 50–78) years. The patients were heavily pretreated with the median number of treatments being 4. There were 5 grade 3–4 toxicities (CMV viremia (2 pts), skin rash (2 pts), and febrile neutropenia (1 pt)). Seven patients had evidence of disease progression and were removed from study. One patient completed the treatments with a mixed response. One patient had a sudden death while on study. The median duration of therapy was 6 weeks (range 3 weeks to 17 weeks). The accrual was stopped after 14 patients when the interim analysis showed a lack of anti-tumor efficacy and significant toxicity. Conclusion: This phase II study in patients with relapsed / refractory myeloma or amyloidosis demonstrated no evidence of clinical activity with use of Campath. In addition it was also associated with significant toxicity in this population. We do not recommend use of campath as a primary treatment modality in these patients. The lack of efficacy despite demonstrated expression of CD52 on clonal plasma cells once again highlights the need for combining treatments with multiple mechanisms in order to improve the response and outcome in these patients. Whether it can synergize with other therapies at lower doses without significant toxicity remains unknown.

Abstract Background: We reported a high response rate with the combination of lenalidomide plus dexamethasone (Rev/Dex) as initial therapy for newly diagnosed multiple myeloma (Blood2005;106:4050–3). We now present new data on time to progression (TTP), progression free survival (PFS), and overall survival (OS) from this phase II trial, and also include updated response data. 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 autologous stem cell transplantation (SCT), 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. Response was assessed by modified EBMT/International Myeloma Working Group Uniform Response criteria. 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. Median follow up is 21 months. Thirteen patients proceeded to SCT following initial therapy with Rev/Dex and were censored at that time point for purposes of calculation of response, TTP and PFS. Patients who discontinued therapy to proceed to SCT received a median of 4 cycles of therapy (range, 4–13), while those staying on Rev/Dex (n=21) received a median of 19 cycles of therapy (range, 2–30). Thirty-one of 34 patients (91%) achieved an objective response to therapy; including 6 patients (18%) achieving a complete response (CR) and 13 patients (38%) achieving very good partial response for a CR+VGPR rate of 56%. The CR+VGPR rate among the 21 patients staying on Rev/Dex as primary therapy without SCT was 67% (CR 24%, VGPR 43%). Median TTP, PFS, and OS have not been reached (Figure). By Kaplan-Meier method, the estimated 2 year progression rate was 18%. The 2-year PFS rate and OS rate were 74% and 91%, respectively. Fifty-five percent of patients experienced grade 3 or higher non-hematologic toxicity at any point during therapy, most commonly fatigue (21%), neutropenia (21%), anxiety (6%), pneumonitis (6%), muscle weakness (6%), and rash (6%). Two patients died on study: one attributed to infection unrelated to therapy, the patient had stopped all therapy for over a month before the fatal infection occurred; the other death was due to infection felt possibly related to therapy. One patient developed a pulmonary embolism (grade 4 toxicity), 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 a high CR+VGPR rate of 56% for the trial, and 67% among the subset of patients receiving this regimen as primary therapy. Responses are durable with a low progression rate at 2 years.

Abstract Introduction: Stem cell transplant is an increasingly widely applied technique for managing amyloidosis. As in multiple myeloma, the treatment would not be expected to be curative and patients would be expected to relapse in most instances. In addition, approximately one-third of patients fail to respond to initial therapy. The role of second transplantation in treatment failures or patients who relapse is reviewed. Patients: Of 242 patients undergoing high dose conditioning and stem cell transplantation for amyloidosis (AL) at the Mayo Clinic, 6 subsequently underwent a second transplant. Results: Patient 1, who had single organ renal amyloid nephrotic syndrome responded and relapsed at 73 months, received a second stem cell transplant achieving a hematologic complete response with normalization of a markedly elevated pre transplant free light chain (59 to 1.9 mg/dL) and is alive at 12 months. Patient 2 relapsed 42 months after transplant for single organ renal amyloidosis; the second stem cell transplant, has normalized his free light chain ratio reduced his urinary protein loss from 5450 to 1482 mg/day and he is alive 21 months following his second transplant (63 months overall). Patient 3 relapsed 34 months after a response of renal and cardiac amyloidosis. After transplant two, he failed to achieve a 50% reduction in his free light chain, began hemodialysis 7 months post transplant and died one year post transplant. Patient 4 with single organ cardiac involvement relapsed 30 months following his first transplant and died on day +5 of his second transplant of an acute pulmonary embolus. Patient 5 was a response failure after his first transplant, had a second transplant 35 months later, having failed Dexamethasone followed by Melphalan/Dexamethasone therapy, and died 3 months later of progressive disease. Patient 6 had single organ vascular involvement manifest by calf and thigh claudication and failed to respond with his first transplant with a persistently elevated free light chain. The second transplant was performed six months after the first without further free light chain reduction and persistent claudication at 11 months. Conclusion: Patients who failed to achieve a hematologic response after a first transplant appear not to benefit from a second course of high dose therapy. Among 4 patients who were previously responsive and relapsed, one achieved a hematologic complete response, one achieved a hematologic partial response, and two died. The two patients achieving a second hematologic response had the longest response duration (73 and 42 months) following their first transplant, and the second response presumably reflects favorable (indolent; non proliferative) biology of the underlying plasma cell dyscrasia. High dose therapy and stem cell transplant may be an effective salvage regimen for patients who responded to a first transplant and whose response lasted greater than 36 months.