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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: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder that carries a 1% per year risk of progression to multiple myeloma (MM) or related malignancy. The prevalence and natural history of MGUS, in which by definition intact immunoglobulin heavy chain (IgH) is expressed, has been well described. However, up to 20% of myeloma (MM) is characterized by complete lack of IgH expression (Light-chain MM); the prevalence of a corresponding precursor entity, light chain MGUS (LC-MGUS) has not been determined. We report the first prevalence estimates of LC-MGUS in the general population from a large, well-defined geographic population using modern laboratory techniques. Methods: The cohort for this study was derived from one previously assembled by us to estimate the prevalence of MGUS (N Engl J Med2006;354:1362-9). The original cohort used to estimate the prevalence of MGUS consisted of 21,463 of the 28,038 enumerated residents aged 50 or over of Olmsted County Minnesota as of January 1, 1995. The sensitive serum free light chain (FLC) assay (The Binding Site Limited, Birmingham, U.K.) was performed on stored serum samples from these 21,463 persons. IgH expression was determined by immunofixation on all FLC results that had an abnormal kappa/lambda ratio (<0.26 or >1.65). LC-MGUS was defined as the presence of an abnormal FLC ratio and a negative immunofixation for IgH expression. Results: Adequate stored serum samples were available in 20,733 (97%) of the 21,463 persons. To date, the FLC assay has been performed and results were available for analysis on samples from 16,637 persons. An abnormal FLC ratio was observed in 572 persons. IgH expression was detected in 255 of these cases on immunofixation; these persons are considered as having MGUS, and were excluded from the estimation of LC-MGUS prevalence. This resulted in 317 persons out of 16,637 who had an abnormal FLC ratio without evidence of IgH expression, resulting in an estimated prevalence of LC- MGUS of 2%. Of the 317 cases of LC-MGUS identified in this study, 217 were kappa and 100 were lambda; in 35 cases the presence of the corresponding monoclonal light chain was apparent on immunofixation. The median age of the cohort of LC-MGUS was 62 years; males=151, females =166. The involved FLC level ranged from 0.118–270.0 mg/dL. The FLC ratio ranged from 0.014–0.253 (lambda) and 1.67–511.01 (kappa). So far, progression to multiple myeloma has occurred in 4 patients, a rate much higher than what is expected based on the prevalence of myeloma in the general population. Two additional patients have developed CLL. Conclusions: LC-MGUS is prevalent in 2% of the general population aged 50 years of age or older. The natural history of this disorder needs to be determined.

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 Due to the diagnostic sensitivity of serum free light chain quantitation for monoclonal light chain diseases, it has been suggested that urine assays no longer need be performed as part of the diagnostic algorithm for monoclonal proteins. We reviewed our experience to determine the relative diagnostic contribution of urine assays. Methods: Patients with a monoclonal gammopathy and monoclonal urinary protein at initial diagnosis who also had a serum immunofixation and serum free light chain quantitation within 30 days of diagnosis were identified (n = 428). The laboratory results for serum protein electrophoresis, serum immunofixation, serum free light chain, urine protein electrophoresis, and urine immunofixation were reviewed. Results: The patients in this cohort had diagnoses of multiple myeloma, primary amyloid, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, solitary plasmacytomas, and other less frequently detected monoclonal gammopathies. By definition of the cohort, all 428 had a monoclonal urine protein. 86% had an abnormal serum free light chain K/L ratio, 81% had an abnormal serum protein electrophoresis, and 94% had an abnormal serum immunofixation. In only 2 patients, however, were all 3 serum assays normal. Both of these were patients with monoclonal gammopathy of undetermined significance (idiopathic Bence Jones proteinuria). Conclusion: Discontinuation of urine studies and reliance on a diagnostic algorithm using solely serum studies (protein electrophoresis, immunofixation, and free light chain quantitation), missed 2 of the 428 monoclonal gammopathies (0.5 %) with urinary monoclonal proteins, and these 2 cases required no medical intervention.

Abstract Background: The measurement of monoclonal (M) protein in the serum and urine is critical for response assessment and disease evaluation in patients with multiple myeloma (MM). The serum free light chain (FLC) assay offers a new and sensitive method of assessing response to therapy. An important question that has not been adequately addressed is the correlation between 24 hour urine M protein levels and serum FLC measurements, and the extent to which response to therapy estimated using the FLC assay correlates with that assessed using the 24 hour urine M protein level. Methods: A total of 2194 sets of data, with simultaneous UPEP and serum FLC measurement, were studied. These included 752 unique patients, with individual patients having 1–23 paired assessments over time. FLC estimation was carried out using the serum FLC assay (Freelite; The Binding Site Limited, UK) performed on a Dade-Behring Nephelometer. Based on the established reference range, kappa/lambda FLC ratio <0.26 or >1.65 were defined as abnormal indicating the presence of monoclonal lambda and kappa FLC, respectively. The monoclonal light chain isotype was considered the involved FLC isotype, and the opposite light chain type as the uninvolved FLC type. The Urine M protein by UPEP was compared to the serum levels of the involved light chain using Spearman Rank Correlation. For comparisons in individual patients over time, those with at least 10 measurements each were studied. Results: The median involved FLC level in patients with an undetectable urine M protein was 2.3 mg/dl compared to 32.2 mg/dL among those with a detectable urine M protein (P<0.001). Among the 1676 points with an abnormal FLC ratio, only 75% had an M protein detected in the urine, P < 0.001. Conversely, among patients with a positive urine M-protein, 91% had an abnormal FLC ratio. When all the 2194 data points were considered together, there was a significant correlation between the urine M protein level and the FLC levels (FLC level calculated as the difference between involved and uninvolved levels), rho=0.763, P < 0.001. The correlation did not change when patients with a serum creatinine of over 2.5 were excluded. The correlation between FLC levels and urinary M protein can be affected by several factors such as renal function that will differ across patients. Therefore, we examined whether the correlation between the two variables is stronger when the variations introduced by inter-patient differences in the relationship between the two variables are eliminated. In order to do this, we studied individual patients on whom multiple data points over time were available. One patient who had the maximum number of paired assessments (23 pairs) of serum FLC level and urinary M protein; the correlation between the two variables over time was highly significant, rho 0.981, p<0.001. Similarly 26 other patients who had measurable urine M protein levels in whom 10 nor more paired observations over time were available, also showed significant correlations, rho, range 0.726–0.981, p<0.01. Conclusion: There is a significant correlation between urine M-protein and serum free light chain across patients and the correlation is stronger in individual patients in whom the effect of inter-patient variation in other confounding factors can be eliminated. These data if confirmed in a clinical trial setting would support the use of serum FLC levels instead of urinary M protein measurements to assess response to therapy.

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.