Giuseppina Palladini

Consensus guidelines for the conduct and reporting of clinical trials in systemic light-chain amyloidosis.

This manuscript summarizes the recommendations that emerged from the first Roundtable on Clinical Research in Immunoglobulin Light-chain Amyloidosis (AL), a meeting sponsored by the Amyloidosis Foundation (Clarkston, MI, USA) to develop a consensus of experts on a modern framework for clinical trial design and drug development in AL. Recent diagnostic and technical advances in AL, and updated consensus guidelines for assessing hematologic and organ responses, enable us to define study populations, appropriate end points, and other criteria for all phases of clinical research. This manuscript provides a framework for the design and conduct of systematic collaborative clinical research in AL to encourage more rapid testing of therapies and to expedite new drug development and approval.

Melphalan and dexamethasone with or without bortezomib in newly diagnosed AL amyloidosis: a matched case-control study on 174 patients.

Oral melphalan and dexamethasone (MDex) is a standard treatment for patients with AL amyloidosis who are not eligible for stem cell transplantation at many referral centers. However, following encouraging reports on the activity of bortezomib combined with alkylators and dexamethasone, these combinations are being moved to frontline therapy. We compared the outcome of 87 patients treated with bortezomib plus MDex (BMDex) with that of 87 controls treated with MDex. Patients and controls were matched for age, cardiac and renal function and free light chain burden. A higher rate of complete responses was observed with BMDex (42 vs 19%), but this did not result in a survival improvement in the overall population. However, a significant survival advantage for BMDex was observed in patients without severe (New York Heart Association class III or IV) heart failure and with N-terminal pro-natriuretic peptide type-B <8500 ng/l. Patients treated with full-dose dexamethasone had similar response rates and survival whether they received bortezomib or not. Intermediate-risk patients who are not fit enough to receive high-dose dexamethasone are likely to take the greatest advantage from the addition of bortezomib to MDex.

Sympathectomy or Doxazosin, But Not Propranolol, Blunt Myocardial Interstitial Fibrosis in Pressure-Overload Hypertrophy

The adaptive changes that develop in the pressure-overloaded left ventricular (LV) myocardium include cardiomyocyte hypertrophy and interstitial fibrosis. Although the former is known to depend to a sizeable extent on sympathetic (over)activity, little information exists whether the same applies to the latter, ie, whether excess catecholamine exposure contributes to the imbalance between collagen deposition by fibroblasts and degradation by matrix metalloproteases (MMPs), eventually leading to LV collagen accumulation. Sprague-Dawley rats were subjected to abdominal aortic banding (B) or sham operation (S) and treated with &bgr;-blockade (Bb, oral propranolol, 40 mg/kg per day), chemical sympathectomy (Sx, 6-hydroxydopamine, 150 mg/kg intraperitoneal twice per week) or vehicle (Vh). Ten weeks later, systolic blood pressure, LV weight, collagen abundance (computer-aided histology), zymographic matrix metalloproteinase (MMP)-2 activity and its specific tissue inhibitor concentration (TIMP-2) were measured. Both sympathectomy and &bgr;-blockade failed to attenuate the banding-induced blood pressure elevation but significantly attenuated the attendant LV hypertrophy. As expected, pressure-overload hypertrophy was associated with interstitial fibrosis (collagen: 4.37±1.23% BVh versus 1.23±0.44% SVh, P<0.05), which was abolished by sympathectomy (2.55±1.31%, P=not significant versus SSx) but left unchanged by &bgr;-blockade (4.11±1.23%, P<0.05 versus both SBb and BSx). &bgr;-blockade, but not sympathectomy, was also associated with an increased TIMP-2/MMP-2 ratio (P<0.05), indicating reduced interstitial collagenolytic activity. In separate groups of banded and sham-operated rats, treatment with the &agr;-receptor blocker doxazosin (10 mg/kg per day) displayed similar antifibrotic and biochemical effects as sympathectomy. Thus in the course of experimental pressure overload, the sympathetic nervous system plays a major pro-fibrotic role, which is mediated via &agr;-adrenergic but not &bgr;-adrenergic receptors.

INTRANUCLEAR DISTRIBUTION, FUNCTION AND FATE OF GLUTATHIONE AND GLUTATHIONE-S-CONJUGATE IN LIVING RAT HEPATOCYTES STUDIED BY FLUORESCENCE MICROSCOPY

The availability of fluorescent probes to detect soluble and protein‐bound thiols has made it possible to investigate some aspects of reduced glutathione (GSH) metabolism and function in intact rat hepatocytes and in hepatocyte nuclei. Monochlorobimane (BmCl) has been employed to study the subcellular compartmentation of GSH and the formation and fate of the BmCl‐GSH conjugate. The occurrence of relatively high concentrations of GSH within the nuclear matrix has been inferred from fluorescence quantitation using image analysis. Concomitant biochemical studies have revealed the presence of a GSH‐stimulated ATP hydrolysis and of an ATP‐stimulated GSH accumulation in isolated nuclei, providing the molecular basis for nuclear glutathione compartmentation. The contemporary use of fluorescent probes to label nuclear free sulfhydryl groups, proteins and chromatin status led to the demonstration that intranuclear accumulation of glutathione may modulate the thiol/disulfide redox status of nuclear proteins and control chromatin compacting and decondensation. Microsc. Res. Tech. 36:243–252, 1997.

Bortezomib in a phase 1 trial for patients with relapsed AL amyloidosis: cardiac responses and overall effects

BACKGROUND Bortezomib is approved for the treatment of multiple myeloma and a role has been suggested in the treatment of systemic AL amyloidosis (AL). METHODS In this phase 1 dose-escalation portion of the first prospective study of single-agent bortezomib in AL, 31 patients with relapsed disease, including 14 (45%) with cardiac involvement, received bortezomib in seven dose cohorts on once-weekly (0.7, 1.0, 1.3, 1.6 mg/m(2)) and twice-weekly (0.7, 1.0, 1.3 mg/m(2)) schedules. Electrocardiographic, Holter and echocardiographic studies were evaluated in all patients to determine safety and response. RESULTS During therapy (median treatment period 210 days), no patient developed significant ventricular or supraventricular rhythm disturbance on 24-h Holter monitoring; however, no patient satisfied study criteria for cardiac response using echocardiographic assessment or New York Heart Association classification. Seven patients (23%) had a ≥ 10% fall in left ventricular ejection fraction, but only one met criteria for cardiac deterioration. The predominant cardiac adverse events were peripheral edema (23%), orthostatic hypotension (13%) and hypotension (10%). Two patients developed grade 3 congestive heart failure, which resolved following treatment interruption. In this Phase 1 portion, the maximum tolerated dose of bortezomib on either schedule was not reached. Hematologic responses occurred in 14 patients (45%), including seven (23%) complete responses. In non-responders mean left ventricular wall thickness increased during the course of treatment. CONCLUSION AL is frequently rapidly progressive; in these patients who had relapsed or progressed following previous conventional therapies, these results suggest that bortezomib may slow the progression of cardiac amyloid with limited toxicity.

The role of minor salivary gland biopsy in the diagnosis of systemic amyloidosis: results of a prospective study in 62 patients.

Histological demonstration of amyloid deposits is required for the diagnosis of amyloidosis. Less invasive approaches than the biopsy of the organs involved, such as abdominal fat and salivary gland biopsy are feasible. In AL amyloidosis, abdominal fat sensitivity is approximately 80%. We report the results of salivary gland biopsy in 62 consecutive patients with suspected systemic amyloidosis and negative abdominal fat aspirates. Amyloid deposits were detected in 7 of the 12 patients in whom amyloidosis was eventually diagnosed. The deposits were characterized as AL l in 4 cases (57%), AL k in 2 (29%), and AA in 1 (14%). In the remaining five patients, amyloidosis was diagnosed by organ biopsy and characterized as AL l in three subjects and AL k in two. Overall, the diagnostic sensitivity of the salivary gland biopsy in patients with negative fat aspirate was 58%, specificity 100% and negative predictive value 91%. A sequential diagnostic approach based on second-step salivary gland biopsy can spare organ biopsy to more than half the patients with systemic amyloidosis. Introduction: Histological demonstration and accurate characterization of amyloid deposit is pivotal in the diagnosis of amyloidosis [1,2]. When amyloidosis is suspected, biopsy of the involved organ could be performed. Although recent patient series do not confirm the previously reported increased risk of bleeding associated with organ biopsy in systemic amyloidoses [3,4], the ready availability of alternative, less invasive, biopsy sites, such as abdominal fat and minor salivary gland, represent appealing alternatives to organ biopsy. In a large series of patients referred for suspected systemic amyloidosis, we recently reported that the sensitivity of abdominal fat is 87% [5]. Thus, approximately 15% of patients with systemic amyloidosis are not diagnosed by abdominal fat aspirate alone. Minor salivary gland biopsy is a simple and safe technique that has been shown to be highly sensitive for the diagnosis of immunoglobulin light chain (AL) amyloidosis [6–9], as well as of familial amyloid polyneuropathy Portuguese type [10]. In this study, we report the results of a sequential approach in patients with suspected systemic amyloidosis and negative abdominal fat aspirate, in whom minor salivary gland biopsy was employed as second step. Methods: Sixty-two consecutive patients referred to the Pavia Amyloidosis Research and Treatment Center between 2002 and 2007 for suspected systemic amyloidosis, in whom amyloid deposit were not detected in the abdominal fat aspirates underwent minor salivary gland biopsy. The patients gave written informed consent according to the Institutional Review Board guidelines. In all subjects, evidence of a plasma cell clone was searched by serum and urine high resolution immunofixation electrophoresis and by the free light chain assay [11]. Mutations related to hereditary systemic amyloidosis were searched by DNA analysis. The salivary gland biopsies were performed in the Department of Rheumatology of the Fondazione IRCCS Policlinico San Matteo, Pavia as previously described [9]. Briefly, after evaluation of the coagulation parameters, minor salivary glands were located by visual inspection and the area disinfected and anesthetized. A small incision was made with a scalpel, and the salivary gland was removed with a biopsy forceps. The sample collected were fixed in formalin and stained with hematoxylin and eosin and Congo red. Congo red stain was performed according to Westermark et al. [12]. Samples deemed positive were analyzed by immuno-electron microscopy [13]. Ultrathin sections were stained with uranyl acetate 5% and lead and scanned with a Philips CM12 electron microscope. To characterize the constitutive protein monoclonal or polyclonal antibodies were employed. In Figure 1, the results of the staining of the salivary biopsy are shown. The diagnosis of amyloidosis was eventually excluded in the remaining patients who were followed for at least 2 years after the salivary gland biopsy or until the cause underlying their clinical syndrome was identified. Results and conclusions: Sixty-two patients were included in the study. Their median age was 62 years (range, 29–79 years) and 68% were males. In 39 patients (63%), a monoclonal component was detected. The monoclonal protein was k in 15 (24%) subjects, l in 21 (34%), and 3 patients (5%) had a biclonal gammopathy. The clinical features leading to suspect systemic amyloidosis in this patient population was nephrotic syndrome in 39 subjects (63%), 18 patients (29%) had an elevated mean left ventricular wall thickness (mLVW), carpal tunnel syndrome and peripheral neuropathy were 80

Circulating Antibodies Recognizing Oxidatively Modified Low-Density Lipoprotein in Children

The occurrence of circulating antibodies (IgG) against oxidatively modified LDL was investigated in a group of normocholesterolemic, healthy, cardiovascular risk-free children and in a comparable group of normal adults. An increased titer of IgG recognizing Cu++-oxidized or malondialdehyde (MDA)-derivatized LDL (MDA-LDL) was a constant feature in children. The antigenic epitopes recognized by these antibodies were generated rather late in the process of copper-mediated LDL oxidation, concomitantly with the formation of fluorescent adducts between reactive aldehydes (including MDA) and apo B100. MDA-LDL was not the only antigen recognized: derivatization of other structurally unrelated proteins, such as HDL, serum albumin, fibrinogen, and transferrin with MDA led to generation of recognizable epitopes. However, among the various modified proteins, LDL exhibited the highest binding activity for IgG present in the group of children. This was associated with an enhanced propensity of LDL isolated from children to undergo in vitro oxidation, despite normal levels of the endogenous antioxidant α-tocopherol. These findings indicate that circulating antibodies recognizing proteins modified with end-products of lipid peroxidation (including LDL) are present in healthy cardiovascular risk-free children. The possibility that LDL oxidation occurs in vivo already in childhood and may act as the real immunogen is an attractive but still unproven hypothesis.

Survival Benefits of Different Antiadrenergic Interventions in Pressure Overload Left Ventricular Hypertrophy/Failure

We observed previously that in rats with aortic banding (Bd), development of left ventricular (LV) hypertrophy is opposed by β-blockade, whereas interventions interfering with α-adrenoceptor function also inhibit interstitial fibrosis. To assess whether these differential structural effects do translate into different effects on LV function and on heart failure mortality, Bd or sham Bd 8-week–old rats were randomized to vehicle treatment (Vh), chemical sympathectomy ([Sx] 6-hydroxydopamine, 150 mg/kg IP twice a week), β-adrenoceptor blockade (propranolol [Pro], 40 mg/kg per day PO), or α-adrenoceptor blockade (doxazosin [Dox], 5 mg/kg per day PO). After monitoring survival for 10 weeks, the survivors were anesthetized to undergo echocardiography and intraarterial blood pressure measurement. Bd-Vh rats showed increased LV and lung weights, as well as LV dilation, depressed endocardial and midwall fractional shortening and a restrictive transmitral diastolic flow velocity pattern. Compared with Bd-Vh rats, all of the actively treated Bd rats showed less LV hypertrophy, LV dilation, and lung congestion but no less depression of midwall fractional shortening. In contrast, Sx and Dox but not Pro treatment were also associated with lesser degrees of diastolic dysfunction and, even more importantly, with a striking increase in survival (sham banded rats, 100%; Bd-Vh, 40%; Bd-Pro, 51%; Bd-Sx, 83%; and Bd-Dox, 82%). Although Pro, Sx, and Dox provide similar midterm protection from development of LV hypertrophy and dysfunction and from circulatory congestion, only Sx and Dox favorably affected mortality. These findings indicate that in the aortic banding rat model, α-adrenoceptors are importantly involved in the pathogenesis of cardiovascular deterioration and disease progression.

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.

Rationale, application and clinical qualification for NT-proBNP as a surrogate end point in pivotal clinical trials in patients with AL amyloidosis

Amyloid light-chain (LC) amyloidosis (AL amyloidosis) is a rare and fatal disease for which there are no approved therapies. In patients with AL amyloidosis, LC aggregates progressively accumulate in organs, resulting in organ failure that is particularly lethal when the heart is involved. A significant obstacle in the development of treatments for patients with AL amyloidosis, as well as for those with any disease that is rare, severe and heterogeneous, has been satisfying traditional clinical trial end points (for example, overall survival or progression-free survival). It is for this reason that many organizations, including the United States Food and Drug Administration through its Safety and Innovation Act Accelerated Approval pathway, have recognized the need for biomarkers as surrogate end points. The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. Underlying this consensus is the demonstration that NT-proBNP is an indicator of cardiac response in all interventional studies in which it has been assessed, despite differences in patient population, treatment type and treatment schedule. Furthermore, NT-proBNP expression is directly modulated by amyloidogenic LC-elicited signal transduction pathways in cardiomyocytes. The use of NT-proBNP will greatly facilitate the development of targeted therapies for AL amyloidosis. Here, we review the data supporting the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC and universally accepted by AL amyloidosis specialists, as a surrogate end point for survival.