Current treatment of AL amyloidosis

Immunoglobulin light chain systemic amyloidosis (AL) is a progressive disease caused by monoclonal light chains with specific mutations that confer a unique propensity to misfold from their native structure to less stable, partially folded intermediates that self-aggregate into oligomers and then into the highly-ordered cross β-sheet structure which defines amyloid fibrils.1 At least 11 additional proteins, synthesized by different organs (liver, intestine, etc) can cause systemic amyloidoses which can be difficult to distinguish from AL amyloidosis on a clinical basis. These proteins form amyloid deposits that share the common tinctorial, green birefringence under polarized light after staining with Congo red, and ultrastructural features, rigid, non-branching fibril with a distinct diameter of 7.5 to 10 nm (Figure 1). The unequivocal identification of the protein forming the amyloid fibril is essential for the choice of therapy.3 A mistake in protein typing may have catastrophic therapeutic consequences, such as performing an autologous stem cell transplant in a patient with transthyretin amyloidosis who should receive a liver transplant. Proteomics technology has significantly improved the typing of amyloid deposits2 and is routinely applied on abdominal fat aspirates at our Center.

Figure 1.Typing amyloid deposits in subcutaneous adipose tissue. The 10–30 mg fine needle fat aspirate can be used for: (A) Congo red staining and analysis under polarized light showing the diagnostic green birefringence (upper left panel, Nikon Eclipse E600, magnification x 200); (B) for typing the amyloid deposits using electron-microscopy immunohistochemistry, in this case the amyloid fibrils are decorated by a gold- labeled antibody recognizing an anti-κ antibody (central upper panel, TEM Philips CM12, magnification x 22000, gold particles are 15 nm in diameter); and for proteomic analysis by bidimensional- PAGE and mass spectrometry. Comparison with control adipose tissue (C1) allowed location of prominent novel spots (squared in green) in patient (C2). Spots corresponding to fragments or post-translational modifications of the amyloidogenic protein were also observed. Identification of the novel spots, and thus amyloid typing, was obtained by MALDI-TOF mass spectrometry and peptide mass fingerprinting.

The amyloidogenic light chains are produced by a bone marrow plasma cell clone usually of limited size, enter the circulation, and target selected organs: heart, kidney, liver, soft tissues and peripheral nervous system, trough specific, but largely undetermined, interactions with local matrix components, such as glycosaminoglycans, and cell membrane constituents. These interactions may promote the formation of light chain oligomers which may represent the main determinant of cell toxicity and the consequent tissue dysfunction. Deposited amyloid fibrils may contribute to organ dysfunction by promoting light chain oligomerization, through increasing macromolecular crowding in the interstitial space,4 and by damaging the tissue architecture. The aim of therapy is the prompt reduction/elimination of the supply of newly formed light chains, that feed the formation of oligomers and fibrils, in order to obtain durable improvement of AL amyloidosis-related organ dysfunction and extend survival.5

Several effective chemotherapy regimens have been developed during the last decade improving significantly the outlook of patients with AL amyloidosis.6

Current treatment of AL amyloidosis

Patients with AL amyloidosis not only have a hematologic malignancy, but also present with progressive dysfunction of one or more organs. A rapid response is essential in order to arrest the progressive organ damage and possibly rescue its function. In addition, the amyloid multiorgan involvement renders these patients particularly susceptible to the toxic effects of chemotherapy. The therapeutic armamentarium has greatly expanded in recent years from melphalan-prednisone as single resource in 19977 to several effective therapies including high-dose dexamethasone-based regimens8,9 combined with melphalan (MDex),10 thalidomide (ThalDex),11 and cyclophosphamide-thalidomide (CTDex),12 high-dose melphalan followed by rescue with autologous stem cell transplantation (SCT),13,14 to the new agents, lenalidomide15,16 and bortezomib.17–19 MDex and SCT are the two most widely used regimens. The French Myeloma Collaborative Group compared these two regimens in a randomized trial and found no significant differences for hematologic or organ responses, and a landmark analysis, examining only patients surviving six or more months showed no survival advantage for SCT.20 Although suboptimal patient selection for SCT may represent a limitation of this study, these results confirmed that MDex has a relevant place in the treatment of AL patients. High-dose melphalan/SCT provides a significant proportion of complete responses which translate into improved quality of life and prolonged survival.21 However, the procedure is toxic for these fragile patients as indicated by the treatment related mortality (TRM) that in the large published series ranges from 11%14 to 27%.22 However, lately a careful patient selection, based also on assessment of cardiac dysfunction using biomarkers,23,24 has significantly reduced the TRM.

Provisional suggestions for choice of chemotherapy in AL amyloidosis

Consensus criteria for best therapy have not yet been established. The choice should be based on tolerability, efficacy and rapidity of action of regimens reported to be effective. The amyloid heart involvement is by far the main prognostic determinant in AL amyloidosis.25,26 The staging system for cardiac amyloidosis employing the biomarkers NT-proBNP26 and troponin27 has been validated in patients undergoing SCT.23

In good risk patients (younger than 65 years, with ≤2 major organs involved, NT-proBNP < 332 ng/L, cardiac troponin T < 0.035 μg/L or cardiac troponin I < 0.1 μg/L,24 creatinine clearance ≥50 mL/min, pulmonary diffusion capacity ≥50% and systolic blood pressure > 90 mmHg) SCT with melphalan 200 mg/m may be considered as front-line therapy. In our clinical practice, this group of patients represents 15% of the whole AL amyloidosis population. With the aim of reducing TRM, a risk-adapted modulation of the dose of melphalan (140–100 mg/m) has been proposed. However, dose reduction translated into reduced hematologic response rate (53%) with a still considerable TRM.28 Adjuvant therapy for patients not achieving CR after SCT has been investigated. Thalidomide and dexamethasone was administered to 31 patients, improving hematologic responses in 42% of them, but was poorly tolerated.29 In a subsequent trial, involving 27 patients, thalidomide was substituted by bortezomib and resulted in improved responses in 7/8 patients.30

Patients at high risk, with advanced cardiac amyloidosis, and increased troponin (cTnI >0.1 μg/L or cTnT >0.035 μg/L) and NT-proBNP (>332 ng/L), and New York Heart Association (NYHA) class III or IV or ECOG performance status ≥3 (not due to polyneuropathy), have a short median survival (3.5 months).31 They represent approximately 20% of all our patients with AL amyloidosis. These patients are in urgent need of an effective therapy, but they are also extremely fragile and sensitive to treatment toxicity, and generally cannot tolerate high-dose dexamethasone. A gentle, rapidly-acting regimen should be preferred. We designed a study addressing specifically treatment of patients with advanced heart failure. Thalidomide was associated with melphalan and attenuated dose of dexamethasone (MTDa) in order to accelerate the response and minimize the corticosteroid toxicity. Out of the 22 patients treated, 6 died due to cardiac amyloidosis before completing cycle 3 and only 8 patients achieved a hematologic response, while 4 obtained durable improvement of cardiac dysfunction.32 Nearly 30% of patients, particularly those who present with reduced ejection fraction at echocardiography, do not survive long enough to have a chance to respond. In these patients, heart transplantation represents the only viable alternative. In subjects with severe heart involvement but preserved ejection fraction, trials on rapidly acting regimens, such as MTDa or CTDa and, possibly, those containing low-dose (0.7–1.0 mg/m) bortezomib, seem warranted.

The bulk (65%) of the patients are at intermediate risk. These patients can benefit from MDex that produced 67% hematologic responses (33% CR) and 48% organ response rate. Responses to MDex resulted in a significant survival benefit and were durable, with complete remissions being maintained for more than three years in 70% of cases.33 CTD represents a viable option and may be particularly indicated in patients who are eligible for SCT but refuse the procedure, and in those in need of a prompt response in consideration of the rapidity of action of this regimen. In this respect, bortezomib, used as single agent19 or in combination with dexam-ethasone17,18 showed high response rates and a remarkable rapidity of action (median time to response less than one month).17 The addition of bortezomib to MDex may combine the rapidity of action with the durability of response. Phase III trials are scheduled to open in the US and Europe in 2009 comparing MDex versus MDex plus bortezomib in newly diagnosed patients with AL not eligible for SCT with 200 mg/m of melphalan. Relapsing and refractory patients may benefit from bortezomib17–19 and lenalidomide15,16 associated with dexamethasone. Whenever possible, all patients should be treated in clinical trials.

Supportive care is fundamental and aimed at maintaining quality of life, delaying organ failure and prolonging survival while specific treatment has time to take effect. Organ transplantation, particularly heart transplantation, can be offered to patients who attain CR but have irreversible organ damage, but can also represent an option to render a patient with end stage organ failure eligible for high-dose chemotherapy.

Amyloid deposits can regress if the supply of the precursor is suppressed

The first histological documentation that amyloid deposits can be reabsorbed if the synthesis of the amyloid precursor is shut down was provided by Henning, father of the great hematologist, Jan Waldenström.34 He observed rapid resolution of hepatomegaly in a matter of a few weeks after successful surgical treatment of lymphoid tuberculosis fistulae with resolution of amyloid deposits at biopsy. In this case, the amyloid deposits were constituted of serum amyloid A (SAA), an acute phase protein synthesized by the liver under stimulation of the proinflammatory cytokines. In chronic tuberculosis infection, persistent high concentration of serum SAA results in formation of amyloid deposits in target organs: kidney, spleen and gastrointestinal tract.

In the present issue, the Groningen group reports the histological regression of amyloid in abdominal fat tissue in 51 patients following chemotherapy.35 They observed a significant reduction of amyloid deposits in 80% of patients 3.2 years after achieving complete response, and no significant reabsorption in 9 patients achieving a partial response. The first consideration is that regression of AL amyloidosis is a slow process.

The molecular bases for the susceptibility of amyloid fibrils to the endogenous clearance machinery are not known but may be related to the structural organization of amyloid fibrils and their capacity to elicit a local cellular/inflammatory response. Intense research activity is ongoing aiming at targeting the amyloid deposits with innovative drugs capable of removing shielding constituents of amyloid deposits, such as serum amyloid P component (SAP)36 and with amyloid-reactive antibodies37 in order to promote and accelerate the reabsorption of amyloid through endogenous clearance.

Another interesting observation is that amyloid regression was not observed in the 9 patients who achieved a partial response. These patients had presented a median concentration of free light chains (FLC) that was more than eight times higher than that of complete responders (1,010 vs. 122 mg/L), had a more advanced disease, and the residual FLC after chemotherapy, although reduced by at least 50% by definition, was still substantial (with a median above 100 mg/L). Therefore, it is not surprising that in this small subgroup of patients, no significant regression of amyloid deposits was observed and that 7 of these patients died mostly because of amyloid progression. These data should not be interpreted such that partial response does not provide any clinical benefit.

A new paradigm for treatment strategy

Partial response may be a potentially misleading concept in AL amyloidosis, a disease caused by increased concentration of misfolded light chains. Clearly, the 50% reduction in concentration of FLC may have very different clinical consequences, based on the absolute concentration of residual FLC. For instance, if the starting FLC concentration is 2,000 mg/L, a residual FLC concentration of 800 mg/L, although fulfilling the criteria for partial response (60% reduction), is expected to continue to exert a substantial tissue toxicity with reduced survival. On the other hand, in a patient with a starting FLC concentration of 100 mg/L a partial hematologic response with residual FLC concentration of 40 mg/L should result in a substantial relief from light chain toxicity and, possibly, obtain a negative balance between amyloid deposition and reabsorption with prolonged survival. In larger series of patients, it has been shown that achieving a partial hematologic response translates into improved overall survival.14,38 However, it has been reported that the percentage of FLC reduction does not predict for survival, but the absolute level of FLC achieved after SCT therapy does.39 Ideally, one should reduce the concentration of FLC below the toxic threshold causing organ dysfunction.

Now, thanks to the availability of sensitive cardiac biomarkers it may be possible to link the extent of the hematologic response, i.e. the concentration of free light chains, to improvement of heart function.

Amyloid heart involvement is by far the most important prognostic factor since it determines the death of most of the patients,25 and NT-proBNP26 and troponins27 are sensitive markers of heart damage caused by the amyloidogenic light chains. It has been observed that the reduction in FLC after therapy translates into a rapid decrease of NT-proBNP and improved survival.40 A collaborative study performed on a total of 200 patients from the databases of the Amyloidosis centers in Pavia, Italy and London, UK who had completed one line of treatment, showed that patients who achieved a partial response associated with a decrease in NT-ProBNP have identical outcomes (survival) to the patients who achieved a complete response.41 These data support a new paradigm in the treatment strategy of AL amyloidosis. Given that the aim of therapy is to extend the survival through the achievement of durable improvement of AL amyloidosis-related organ function, chemotherapy should be guided by frequent (every 2 cycles) assessment of FLC and cardiac biomarkers in order to optimize the benefit/toxicity ratio exposing the patients to the lowest, most effective, chemotherapy burden.

Future perspectives

Amyloidogenic plasma cells present an important distinctive feature, compared to myeloma plasma cells: they synthesize a misfolded light chain. The misfolded protein may increase the proteosomal workload and render the cells particularly vulnerable to proteasome inhibition.42 It has been reported that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of myeloma plasma cells to proteasome inhibitors.43 Studies are now ongoing in amyloidogenic plasma cells and clarification of the molecular mechanisms of proteotoxic stress will contribute to develop targeted therapies for AL amyloidosis. The possibility of predicting the response to proteasome inhibitors,43 allows the prompt institution of alternative treatments in patients who are unlikely to respond to this class of drugs. Furthermore, the development of new drugs targeting other components of the ubiquitin-proteasome system,44 besides the proteasome, may offer new therapeutic opportunities, including combination therapies.

Although great advancements have been made in understanding the molecular basis of protein misfolding and fibril formation, the molecular mechanisms underlying the light chain targeting of specific tissues, such as the heart, and the consequent organ dysfunction, remain elusive. Intensive research is now focusing on this fundamental process, the identification of the molecules involved may lead to a more comprehensive treatment approach of this complex, but possibly curable disease.

Footnotes

• Dr. Palladini is a lecturer at the University of Pavia, and an attending physician at the Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. Dr. Merlini is a professor of Clinical Biochemistry at the University of Pavia, and the director of the Amyloidosis Research and Treatment Center.
• Dr. Merlini received an honorarium from Millennium Pharmaceuticals. No other potential conflict of interest relevant to this article was reported.
• The authors’ research on amyloidosis is supported by Fondazione Cariplo (Milano, Italy; Nobel Project, Transcriptomics and Proteomic Approaches to Diseases of High Sociomedical Impact: A Technology-Integrated Network), the EURAMY project (European Community’s Sixth Framework Program; Ricerca Finalizzata Malattie Rare, Ministero della Salute, Istituto Superiore di Sanità (526D/63); MIUR-PRIN project 2007AE8FX2_003.