Seeking confidence in the diagnosis of systemic AL (Ig light-chain) amyloidosis: patients can have both monoclonal gammopathies and hereditary amyloid proteins

In the report of the first autologous stem cell transplantation (SCT) trial for systemic Ig light-chain (AL) amyloidosis, the authors wrote that patients with hereditary amyloidosis “are never candidates for dose-intensive melphalan.”^1p3669 In hereditary amyloid, the mutant protein is often hepatic in origin, and the standard treatment is liver transplantation, not high-dose chemotherapy.^2-4  In AL, the precursor protein is an immunoglobulin light chain produced by clonal plasma cells, and standard treatment is cytoreductive chemotherapy.^5-7 

The issue of misdiagnosis of AL has been raised by several investigators.^8,9  The national amyloidosis center in the United Kingdom reported that, of 350 patients thought to have AL, 10% had hereditary variants instead, including patients who had understandably failed SCT. The investigators noted that hereditary variants have variable penetrance, making family history an ineffective screen, and that the immunohistochemical (IHC) staining techniques used to type tissue amyloid as derived from Ig light chains were unreliable, although IHC staining for non-Ig amyloid-forming proteins such as transthyretin (TTR) or fibrinogen Aα may be useful.⁹  The lack of reliable staining for all types of amyloid limits the utility of IHC approaches.¹⁰ 

Most hereditary variants are due to point mutations causing amino acid replacements. In the series of 350 patients from the United Kingdom, the identification of hereditary variants was based on polymerase chain reaction (PCR) amplification and sequencing of potentially mutated genes.⁹  Obviously the issue of diagnostic confidence would be moot if the amyloid protein in each case could be extracted easily from tissue and evaluated for identity.¹¹  Currently, such techniques are not practical because, given the variability of deposits, obtaining enough tissue for protein extraction or for immunogold electronmicroscopy is problematic.^11,12  In this study, we asked how often patients with symptomatic amyloidosis might have both a monoclonal gammopathy and a hereditary variant, employing a screening algorithm based on patterns of presentation of hereditary and AL amyloidosis. Our results support the need for routine DNA-based screening and for new methods for typing amyloid.

Between June 1, 2002, and August 1, 2005, patients referred for assessment of systemic amyloidosis were evaluated for monoclonal gammopathy and organ involvement with amyloid as previously described.^13-15  Approval was obtained from the institutional review board of the Memorial Sloan-Kettering Cancer Center for these studies and patients gave written informed consent. We screened all patients in the following categories for hereditary variants whether or not they had a monoclonal gammopathy: (1) African Americans were screened for the presence of a mutant transthyretin (the Val122Ile variant of TTR occurs in 4% of African Americans¹⁶ ); (2) patients with dominant peripheral nervous system involvement were screened for the variants shown in Table 1 (peripheral neuropathy is a common presentation of AL amyloidosis and several hereditary variants¹³ ); (3) patients with isolated renal amyloidosis and no amyloid in the bone marrow were screened for the fibrinogen Aα variant that occurred in 5% of patients from the United Kingdom, all with renal but not marrow amyloid⁹ ; and (4) patients sent for hereditary screening or with a biopsy reporting amyloidosis were screened for all variants shown in Table 1 and tested for a monoclonal gammopathy; some were rebiopsied.

Genomic DNA was extracted from mononuclear cells as previously described.¹⁷  Primer pairs were designed for transthyretin, apolipoprotein AI, apolipoprotein AII, fibrinogen Aα, and lysozyme (Table 1).^3,18-21  PCR amplicons were sequenced at our core facility and results scanned with Chromas Version 1.45 (Griffith University, Queensland, Australia) and evaluated by BLAST (Genbank).

Tissue sections stained for the identification of amyloidosis by Congo red dichroism were also stained for TTR in patients with both a monoclonal gammopathy and hereditary TTR variants and in patients suspected of hereditary disease or senile cardiac amyloidosis, a diagnosis that requires TTR-positive endomyocardial amyloid and wild-type TTR gene sequence.²² 

Polyclonal rabbit anti–human TTR (DAKO, Carpenteria, CA) was used with citric acid pH 6.00 at a 1:2000 dilution. Slides were incubated with the primary antibody overnight at 4°C in a humidity chamber. Secondary antibody at 1:500 dilution in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) was applied for 1 hour at room temperature then washed in PBS. Peroxidase-conjugated tertiary antibody (DAKO) at 1:500 in 1% BSA/PBS was applied for 45 to 60 minutes at room temperature, then washed. Slides were transferred to a diaminobenzidine bath for 5 to 15 minutes, washed in tap water, then counterstained using Harris modified hematoxylin, decolorized with 1% acid alcohol in ammonia water, dehydrated 3 times in 95% ethanol, 100% ethanol, and xylene for 2 minutes each, and coverslipped with mounting media.

Amyloid deposits were TTR-positive if the TTR immunostain specifically demonstrated concordance with the Congo red stained section—that is, if the areas of amyloid by Congo red dichroisim were TTR-positive and the Congo red–negative areas were TTR-negative.

One hundred and seventy-eight consecutive patients were evaluated for amyloidosis and the diagnosis was confirmed in 96%, most of whom were symptomatic (Table 2). In an attempt to determine how often both a monoclonal gammopathy and a hereditary variant occurred in the same patient, patients in 4 categories were screened and 1 patient with both proteins was identified in 3 of the 4 categories. They represented 6% of those screened and 2% of symptomatic patients. One was African-American, one had peripheral neuropathy, and the third was referred for evaluation of hereditary amyloidosis and was diagnosed with Durie-Salmon stage I multiple myeloma. All had bona fide monoclonal gammopathies, not faint bands on urine immunofixation, and all had a variant TTR (Table 2). TTR staining indicated that in 2 cases amyloid was likely due to variant TTR and in one it was not (Table 2).

The amyloidoses are diseases caused by protein misfolding.^23,24  The most common type encountered by hematologists is due to monoclonal immunoglobulin light chains (AL) and can be difficult to diagnose and treat in a timely fashion.¹³  The median survival even with the most aggressive therapy is less than 5 years.⁶  Diagnostic confidence is critical in order to plan therapy, and hereditary and senile cardiac variants are not treated with cytotoxic therapies or SCT.

Our results speak to the need for reliable tools to aid in the diagnosis and management of patients with amyloidosis. Combined with the recent report from the United Kingdom, the data indicate that the issue of diagnostic confidence is important because 1 patient can have both a monoclonal gammopathy and a hereditary variant, representing 2 possible sources of amyloid-forming proteins.⁹  It should also be recalled that the incidence of monoclonal gammopathy of undetermined significance (MGUS) increases with age and that hereditary variants in the United States usually present in older patients. Both MGUS and Val122Ile mutant TTR are also more common in African Americans.

Given the implications for patients and their families if AL is misdiagnosed and a hereditary mutation not identified, we agree with our colleagues in the United Kingdom that all new cases of amyloidosis should be screened for both AL and hereditary variants.²³  The PCR technique we used is reliable and easily implemented in human genetics laboratories. In addition, innovative and improved techniques are needed for typing amyloid from tissue biopsies.^10-12  Finally, the issue of diagnostic confidence becomes even more critical as new therapies are evaluated for both AL and hereditary disease.^8,25 

We thank the many amyloid patients who contributed to this work. We also thank Dr Stephen D. Nimer for continued help and encouragement, the MSKCC Hematology-Oncology fellows for caring for our patients, Joanne Santorsa, RN, for assistance in bone marrow studies, and Dr Kenneth Offit and colleagues in the Department of Human Genetics at MSKCC for their encouragement.