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Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

The combination of oral melphalan and dexamethasone is considered standard therapy for patients with light-chain amyloidosis ineligible for autologous stem cell transplantation. However, previous trials reported different rates of response and survival, mainly because of the different proportions of high-risk patients. In the present study, including a total of 259 subjects, we treated 119 patients with full-dose melphalan and dexamethasone (dexamethasone 40 mg days 1-4), and 140 patients with advanced cardiac disease with an attenuated dexamethasone schedule (20 mg). Hematologic response rates were 76% in the full-dose group and 51% in the patients receiving the attenuated schedule; the corresponding complete response rates were 31% and 12%, respectively. The median survival was 7.4 years in the full-dose group and 20 months in the attenuated-dose group. Use of high-dose dexamethasone, amino-terminal pro-natriuretic peptide type-B >1800 ng/L, a difference between involved and uninvolved free light chains of >180 mg/L, troponin I >0.07 ng/mL, and response to therapy were independent prognostic determinants. In relapsed/refractory subjects bortezomib combinations granted high hematologic response rates (79% and 63%, respectively), proving the most effective rescue treatment after melphalan and dexamethasone. In summary, melphalan plus dexamethasone was highly effective with minimal toxicity, confirming its central role in the treatment of AL amyloidosis. Future randomized trials will clarify whether bortezomib is best used in frontline combination with melphalan and dexamethasone or as rescue treatment.

Immune-modulatory drugs are active in immunoglobulin light-chain amyloidosis and the addition of alkylating agents can potentiate their action. In this phase II prospective trial we used cyclophosphamide, lenalidomide and dexamethasone in the treatment of 21 patients who were refractory (n=13, 62%) or relapsed (n=8, 38%) after prior treatment including melphalan in all cases, bortezomib in 4 and thalidomide in 6. Median number of cycles administered was 4 (range 2-9 cycles). Severe adverse events were observed in 57% of patients, most common being neutropenia (29%). The hematologic response rate was 62%, with one complete response and 5 very good partial responses. Overall median survival was three years. The achievement of CR/VGPR was associated with a significant survival advantage. The combination of cyclophosphamide, lenalidomide and dexamethasone is an effective treatment for relapsed/refractory AL amyloidosis, and good quality hematologic response should be the aim of treatment in this setting. (clinicaltrials.gov identifier: NCT00607581).

Background and Objectives: High-dose melphalan with autologous peripheral blood stem cell transplantation (ASCT) is an effective treatment for systemic primary amyloidosis. This procedure is, however, associated with substantial toxicity and mortality, particularly if the heart is involved. Refined selection of patients suitable for transplantation and personalized adaptation of the doses of melphalan might improve the outcome., Design and Methods: Twenty-two consecutive patients were selected for age, number of organ systems involved, heart and kidney function, and treated with risk-adapted melphalan conditioning. This was first-line therapy in 81% of cases., Results: Fifty-five percent of the patients had amyloid involvement of two organ systems, with renal involvement predominant in half. Approximately 70% received full-dose melphalan. Toxicity was manageable and three transplant-related deaths (14%) occurred only in the early phase of the study. The median overall survival was 68 months. The intent-to-treat hematologic response rate was 55% at +12 months (complete, 36%; partial, 19%), which was accompanied by organ responses in 75%. Survival was positively influenced by: (i) hematologic response at +3 months (complete+partial responses 55%, median not reached, more than 108 months; no response, median 17 months) (p=0.001); (ii) amyloid involvement of a single organ system (p=0.016). Prolonged follow-up demonstrated that remissions are durable, but relapses may occur as 4 of 12 responsive patients (33%) relapsed, three from complete response, between +30 to +38 months., Interpretation and Conclusions: The present risk-adapted approach produced acceptable toxicity and peri-transplant mortality with prolonged survival in responsive patients. Additional therapy should be considered if no hematologic response is observed at +3 months after ASCT.

Waldenström's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma which produces monoclonal immunoglobulin M (IgM). Over the last decade, new treatment modalites have been developed for the management of this disorder. Our objective is to provide treatment recommendations for WM. A review of published reports was facilitated by a MEDLINE computer search and by a manual search of Index Medicus. Other sources included abstracts and conference proceedings. Most patients with WM who are diagnosed by chance without symptoms should not be treated. Initiation of treatment should not be based on level of serum monoclonal protein per se. The presence of cytopenia, significant adenopathy or organomegaly, symptomatic hyperviscosity, severe neuropathy or cryoglobulinemia indicates the need for treatment. The main choices for primary treatment of symptomatic patients with WM include alkylating agents, the nucleoside analogs fludarabine or cladribine and the monoclonal antibody rituximab or combinations of these programs. There are no data from prospective randomized studies to recommend the use of one program over another. Nevertheless, the need for rapid disease control may favor the use of nucleoside analogs, whereas the presence of significant cytopenia may favor rituximab. High dose therapy with autologous stem cell transplantation may induce responses even in patients with resistance to all three class of agents. It may be prudent to avoid nucleoside analogs in patients who are candidates for high dose therapy. Despite the lack of randomized trials, a rational approach to the treatment of patients with WM is possible. Several factors, including the presence of cytopenias, need for rapid disease control, candidacy for autologous stem cell transplantation, age and co-morbid conditions, should be taken into consideration when choosing the most appropriate primary treatment.

Background and Objective: The bleeding manifestations frequently observed in patients with immunoglobulin light chain amyloidosis (AL) have been attributed to different pathogenetic factors: amyloid deposits in several organs and systems leading to failures of these latter, the affinity of amyloid for some clotting factors, and the presence of plasma components interfering with fibrin formation could all induce alterations of clotting tests. This investigation was aimed at defining the prevalence of clotting abnormalities and their clinical manifestations in patients with AL., Design and Methods: Thirty-six consecutive patients with biopsy proven amyloidosis and documented monoclonal gammapathy were enrolled within one year. The following clotting tests were considered in the study: activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), Russell's viper venom time (RVTT), fibrinogen, factor X and alpha-2 antiplasmin., Results: Hemorrhagic manifestations were mild to moderate in nine patients, but severe and untractable in one. The most frequent clotting anomaly was defective fibrinogen conversion to fibrin, as demonstrated by prolongation of both TT (85% of cases) and RT (90% of cases). Low levels of factor X activity were observed in about 1 out of 4 samples, while fibrinogen and alpha2 antiplasmin levels were distributed over a wide range of values. PT was prolonged in 8 and aPTT in 25 patients. The search for lupus anticoagulant was negative in samples showing a prolongation of aPTT and/or RVVT., Interpretation and Conclusions: The prolongation of TT and RT is not dependent on either the presence of a heparin-like substance in the plasma or on fibrinogen levels; furthermore, the prolongation of RVVT is not related to factor X level. The hypothesized presence in the plasma of an inhibitor of fibrin formation could also affect factor X activation by Russell viper venom. The prolongation of TT and RT represents a peculiar feature of amyloidosis. The variability in the behavior of the other clotting times and hemostatic factors studied is mirrored in the heterogeneity of the clinical features observed in this disease.

Background and Objective: Primary amyloidosis is a lethal form of plasma cell (PC) dyscrasia characterized by deposits of monoclonal immunoglobulin light chains that cause organ dysfunction. In contrast to multiple myeloma, the amyloid clone is typically indolent and of small size, and marrow PC clonality is not always apparent. This is generally investigated by analyzing the light chain isotype ratio in bone marrow PC. We investigated whether the degree of PC infiltration (PC%) and clonality (PC isotype ratio) affected survival in 56 consecutive patients with primary amyloidosis., Design and Methods: PC% was determined by morphologic examination. Immunofluorescence microscopy was used to determine the PC light chain isotype ratio. Statistical analysis was carried out using Cox regression models., Results: The degrees of PC clonality and infiltration were inversely correlated with survival (PC isotype ratio, p = 0.001; PC%, p = 0.008). The two variables were weakly correlated (p = 0.02; r = 0.3). Bone marrow PC isotype ratio demonstrated a powerful independent prognostic value at multivariate analysis when analyzed together with congestive heart failure (the major known negative prognostic factor) and PC%. k/l ratio cut-off values of 0.2 (l patients, p = 0.022) and 16 (k patients, p = 0.03) discriminated two groups with a similar number of patients and significantly different survivals., Interpretation and Conclusions: PC clonality and marrow infiltration are important parameters that influence prognosis, presumably because they reflect the amount of pathogenic light chain synthesis.

Background and Methods: Familial occurrence of immunoglobulin-related (AL) amyloidosis has occasionally been reported. In this work we describe the concomitance of systemic amyloidosis and monoclonal gammopathy (one case of Waldenström's macroglobulinemia and two cases without multiple myeloma or related diseases) in three Italian siblings, two males and one female., Results and Conclusions: All of them showed a common pattern of polyneuropathy to different degrees; two presented a sicca syndrome and one also suffered from nephropathy. Two of them showed the same HLA typing with the same light chain type (k), but had different presenting symptoms. Polyneuropathy and a history of peptic disease in two cases was suggestive of type III familial amyloidotic polyneuropathy (FAP) occurring in the setting of a familial monoclonal component. However, immunohistochemical studies on different tissue specimens using anti-apolipoprotein A1 and anti-transthyretin antibodies were negative. Further screening of DNA samples for transthyretin (TTR) gene mutations was also negative. Clinical and laboratory investigations ruled out reactive or senile amyloidosis and immunohistochemical studies with anti-light chain antibodies on amyloidotic tissue specimens were positive. As a consequence, this family represents a new case of familial AL-amyloidosis.

Background: Sporadic observations suggest a possible taste impairment in AL amyloidosis, but the frequency and intensity of this sensory anomaly are not known., Materials and Methods: We submitted 21 AL amyloidotic patients, drawn from subjects referred to the Amyloidosis Study Center of the Department of Internal Medicine and Medical Therapy, University of Pavia, to suprathreshold scaling analysis., Results: Taste acuity was reduced in most of them. Every taste showed independent behavior, and 90% of these patients were hypogeusic. True ageusia for one or two tastes was observed in 35% of patients. No patient was aware of reduced taste acuity. Macroglossia did not seem to play a prominent role in dulling tastes., Conclusions: Impairment of taste perception suggests that gustative neuropathy is a frequently unnoticed expression of sensory involvement in AL amyloidosis.

Background: Routine electrophoretic analysis fails to detect a monoclonal component (MC) in a considerable portion of AL amyloidosis patients. We investigated whether the combination of immunofixation (IF) on agarose gel electrophoresis and bone marrow plasma cell (BMPC) light chain kappa/lambda ratio analysis could contribute to diagnosis in these cases. The possible use of the BMPC kappa/lambda ratio in monitoring the clone was also investigated., Methods: We performed BMPC kappa/lambda ratio analysis and IF of serum and urine in 16 selected patients with no detectable MC at routine analysis, despite clinical features suggestive of primary amyloidosis. An anti-idiotypic monoclonal antibody specific for the amyloidogenic immunoglobulin and the BMPC kappa/lambda ratio were used to monitor the clone in a patient who underwent autologous peripheral blood stem cell transplantation., Results: Abnormal kappa/lambda ratios were found in 14 (sensitivity 87.5%), and a MC in 12 (sensitivity 75%). Combination of the two analyses confirmed diagnosis in all cases. In one patient changes in the size of the clone, monitored on serial bone marrow aspirates by an anti-idiotypic antibody, paralleled variations of the kappa/lambda ratio., Conclusions: This study demonstrates that the combined use of IF and the BMPC kappa/lambda ratio is extremely powerful in AL amyloidosis. In addition, the BMPC kappa/lambda ratio should be considered for monitoring the amyloidogenic clone when serum or urine MC is not quantifiable.

There is increasing evidence that monoclonal proteins are implicated in the development of peripheral neuropathy. Approximately ten percent of patients with peripheral neuropathy of unknown cause have a monoclonal protein and this rate is significantly higher than prevalence rates of monoclonal protein in comparable segments of the general population. Extensive clinical, electrophysiological and immunopathological evidences indicate that peripheral neuropathy associated with monoclonal protein are heterogeneous, including: 1. the demyelinating, predominantly sensory neuropathies associated with anti-MAG antibodies; 2. the axonal, sensory neuropathies associated with anti-sulfatide and anti-chondroitin sulfate antibodies; 3. the motor neuropathies associated with anti-GM1 antibodies. Patients with chronic polyneuropathies should be evaluated for underlying plasma cell dyscrasia.

Background: Inflammatory cytokines released by either the neoplastic or reactive cells in Hodgkin's disease (HD) might mediate its peculiar clinical and histopathological features. We investigated by Northern blotting the gene expressions of the pyrogenic and inflammation-associated cytokines IL-1 alpha, IL-1 beta, TNF-alpha, TNF-beta (lymphotoxin) and IL-6 in 14 HD lymph nodes and studied their relation to systemic symptoms (B symptoms)., Methods: Two ug of poly(A)+RNA from 14 HD lymph nodes (8 from symptomatic and 6 from asymptomatic patients, of different histological type and disease stage) were subjected to agarose electrophoresis, Northern blotted and hybridized to the various cytokine cDNA probes., Results: The inflammatory cytokines were expressed very heterogenously in HD, even in lymph nodes with the same histological type and with similar stromal inflammatory reactions. IL-1 beta was increased about 2 to 10 times in 5 of 8 lymph nodes from patients with B symptoms, whereas the other cytokines were heterogenously expressed in both symptomatic and asymptomatic patients. Statistical analysis on densitometric values demonstrated that the difference in IL-1 beta expression between symptomatic and asymptomatic patients was significant (p less than 0.02)., Conclusions: These results support the hypothesis of increased IL-1 levels in tumoral lymph nodes from symptomatic HD patients.