AL-Amyloidosis, g Light Chain-Related Guillermo A. Herrera St. Louis University St. Louis, MO

Case History: Fifty-two year old female with history of hypertension on treatment, fibrocystic breast disease and carpal tunnel syndrome presented with nephrotic-range proteinuria (6.5 grams per 24 hour collection). Physical examination was only remarkable for lower extremity edema. Collagen vascular disease work-up was negative. Laboratory data revealed no evidence of anemia, a BUN of 8 mg/dl and a serum creatinine of 0.7 mg/dL. CBC was normal. A renal biopsy was performed. Renal Biopsy Results Click to view with ImageScope Click to view with a Web-Based Viewer Light Microscopy: Ten glomeruli were identified in the specimen submitted for light microscopic examination. In the glomeruli, amorphous, eosinophilic, PAS positive hyaline material was identified in mesangial areas extending into peripheral capillary walls. As a result of the mesangial expansion, the capillary spaces appeared compressed. The normal mesangial matrix was replaced by the above mentioned material which was Congo Red and Thioflavin T positive. Apple green birefringence was demonstrated in association with the Congo Red staining areas. Silver positive spikes were noted protruding laterally from the replaced mesangial areas. No segmentally or globally sclerosed glomeruli were present. Trichrome stain showed focal, mild fibrosis associated with tubular atrophy and drop out. Desquamation, fragmentation and vacuolization of proximal tubular cells was a prominent finding. A few tubular casts were present but there were no fracture planes in these casts and no reaction of the surrounding interstitium was noted. Mild thickening of the walls of the arterioles and small sized arteries was additionally identified. Thioflavin T stain also revealed punctuate fluorescence in the arteriolar walls and Congo Red stain showed focal apple green birefringence in small vessel walls. Immunofluorescence: Six glomeruli were seen in the specimen submitted for immunofluorescence studies. Stains for IgG, IgA, IgM, C3, Clq, fibrinogen, albumin and kappa were negative, as well as the auto-control section in glomeruli. There was distinctive mesangial and peripheral capillary wall fluorescence for g light chains. There was also granular staining in proximal tubules for light chains, predominantly l. Punctate fluorescence was identified along arteriolar and small arterial walls for llight chains. There was no staining in the interstitium for any of the immunoreactants, including g light chain. The auto-control section was negative in tubular interstitial and vascular compartments. Electron Microscopy: Ultrastructurally, there was diffuse, generalized effacement of the foot processes of the visceral epithelial cells. Aggregates of randomly distributed, non-branching, 8 to 10 mm in diameter fibrils were identified in subepithelial locations and mesangial areas replacing the normal mesangial matrix. Along the peripheral capillary walls there was evidence of basement membrane reaction around the fibrillary aggregates and the same fibrils deposited in the mesangium resulted in marked expansion and compression of adjacent capillary spaces. Vacuolization, fragmentation and desquamation of proximal tubular cells was a prominent finding. In addition, the lysosomal system in the proximal tubules appeared prominent with occasional large and atypical lysosomes noted. No immune complex deposits were identified in any location. Careful evaluation of the few vessels present in the specimen showed focal deposits of fibrillary material similar to those previously described. Diagnosis: AL-Amyloidosis, g light chain-related. Post Renal Biopsy Clinical Course: After the diagnosis of AL-amyloidosis was made in the renal biopsy, a serum protein electrophoresis revealed low IgG and IgM and normal IgA but no monoclonal spikes. Urine protein electrophoresis demonstrated freeg light chains. A bone marrow aspirate and biopsy revealed 15% plasma cells with atypical plasma cells confirming the diagnosis of an associated plasma cell myeloma. The patient was treated with 4 courses of Melphalan and Prednisone with no response. Her urine protein excretion actually increased to 8 grams in 24 hours collection and the amount of urinary free g light chains also increased. Her treatment protocol was then changed to VAD (Vincristine, Adriamycin and Doxarubicin) one year and 8 months after the initial treatment was instituted. She remained refractory to therapy and renal failure ensued. The patient died two and a half years after the diagnosis of AL-amyloidosis was made. Discussion This patient presented with nephrotic syndrome and normal renal function. A renal biopsy was performed to determine the cause of the nephritic syndrome and a diagnosis of amyloidosis is established. The demonstration of light chain monoclonality (l) in association with the amyloid deposits established a diagnosis of primary (AL) amyloidosis. Further work-up uncovered a plasma cell dyscrasia and the patient received treatment using two different chemotherapeutic protocols with poor results. Clinical and Pathologic Reatures of Al-Amyloidosis The annual incidence of AL-amyloidosis has been estimated to be 1 per 100,000 individuals with up to about 2000 new cases diagnosed each year. This disease usually manifests during the 5th to the 6th decade and has a male predominance. Although serum protein electrophoresis is a good screening test for patients with plasma cell dyscrasias, light chain secreting and non-secreting myeloma patients generally lack a monoclonal spike in the serum. In a study of 229 patients with AL-amyloidosis in only 40% a monoclonal spike was detected by serum protein electrophoresis while in 68% of these patients the monoclonal protein was detected by immunoelectrophoresis which is a more sensitive technique. Because light chains are freely filtered through the glomeruli, examination of the urine for Bence Jones proteinuria using immunoelectrophoresis and/or immunofixation is important to support the diagnosis. The urine may have to be significantly concentrated before a monoclonal spike can be detected. Immunofixation electrophoresis is the most sensitive method to detect monoclonal proteins in serum and urine samples. A newer effective method to confirm a diagnosis of plasma cell dyscrasia is to look for free light chains in the serum. Due to the relative rarity of the disorder and the non-specificity of the clinical manifestations, AL-amyloidosis is often not recognized as the cause of renal dysfunction until late in the course of the disease. If there are signs and symptoms suggesting amyloidosis, gingival, rectal or abdominal pad biopsies may be used for diagnostic purposes. However, currently the diagnosis of AL-amyloidosis is often made by nephropathologists by demonstrating the presence of amyloidosis in association with monotypical light chain deposition. g VI represents the most common light chaintype in these cases. In some cases the pathologist must have a high degree of suspicion to make the correct diagnosis. The amyloid deposits may be subtle and difficult to confirm using a Congo Red stain which remains the preferred tinctorial approach to diagnose amyloid. On the contrary, Thioflavin T stain is much more sensitive and can depict very small amounts of amyloid. Ultrastructural examination can confirm the diagnosis but occasionally the amyloid deposition is only focal and segmental. The affected mesangial areas may not be present in the sample submitted for EM or difficult to find. Ultrastructural immunolabeling may be helpful in these circumstances by highlighting these scattered amyloid deposits. Ultrastructural criteria for diagnosing amyloid must be strict. When mesangial matrix is examined at high magnification it may mimic amyloid creating a possible source of confusion. In fibrillary glomerulopathy the fibrils are much thicker in diameter (approximately 15-20 nm) than amyloid. Collagen and pre-collagen must also be considered in the differential diagnosis of amyloidosis by EM; the periodicity of collagen and parallel disposition of precollagen fibrils are of help in establishing the correct diagnosis. Also in amyloidosis the fibrils colocalize with serum amyloid-P component. Amyloid deposition can occur in any of the three renal compartments. Vascular amyloid deposition is usually accompanied by glomerular amyloidosis but there are rare cases in which only vascular AL-amyloidosis has been demonstrated. Amyloid deposition restricted to the interstitium has not been documented in the literature. An important challenge to the recognition of AL-amyloidosis is the fact that the antibodies to light chains employed in the routine battery of immunofluorescence stains used to evaluate renal biopsies do not recognize all abnormal light chain proteins; therefore, the amyloid deposits may not stain for kappa or g light chains suggesting non-AL amyloidosis. Furthermore, the heavy chain (AL) amyloidosis, although apparently quite rare, also represents a challenge in diagnosis. A significant number of cases with AL-amyloidosis at the time of initial diagnosis fail to show evidence of a plasma cell dyscrasia if the bone marrow is examined using routine morphologic techniques. To identify the underlying plasma cell dyscrasia it is recommended that bone marrow aspirates be examined by flow cytometry and/or immunohistochemistry to detect he clonal population of plasma cells. A normal number of plasma cells in the bone marrow does not rule out a diagnosis of myeloma. The fact that the kidneys are being affected by amyloidogenic light chains is viewed by most as indirect evidence that there must be an underlying malignant plasma cell disorder. It remains the responsibility of the pathologist to document that such malignant process is present. Some oncologists are still reluctant to treat patients with AL-amyloidosis unless unequivocal evidence of myeloma (plasma cell dyscrasia) is found in the bone marrow specimen, a position that is becoming untenable. Pathogenesis: There have been a number of recent advances in the understanding of the pathogenesis of AL-amyloidosis. Major efforts have been directed towards identifying the primary structural features (amino acid sequences) that differentiate amyloidogenic from non-amyloidogenic light chains. An in vitro system of renal amyloidosis has provided very useful information regarding how mesangial cells interact with monotypical light chains and produce amyloid. Amyloidogenic light chains interact with a yet not fully characterized receptor on the surface of the mesangial cells. When mesangial cells are incubated with amyloid producing glomerulopathic light chains a phenotypic change into a macrophage lineage has been shown to occur. Furthermore, internalization of light chains through a clathrin-mediated mechanism and lysosomal processing have been demonstrated to play important roles in the process of amyloid formation. Delivery of amyloidogenic light chains to the mature lysosomal system is a crucial and integral component of the process of amyloidogenesis. The in vitro model has depicted crucial steps in this process amenable to possible therapeutic intervention. The existing in vitro as well as the availability of in vivo experimental models that duplicate the human form of AL-amyloidosis will provide further insight into the cause and, ultimately, the effective prevention and treatment of this relentless disease process. Treatment, Prognosis and Future Expectations The treatment of patients with AL amyloidosis remains limited. The major therapeutic efforts are at reducing the amyloid protein precursor-light chains by administration of chemotherapeutic agents such as Melphalan and Prednisone. Although control of the plasma cell clone can be achieved, the disease process is rarely reversible and most often renal deterioration occurs. In one randomized prospective study treatment with these drugs reduced mortality in a group of patients with amyloidosis compared to a control group receiving Colchicine alone; however, the progression of the renal disease and incidence of end stage renal disease was not reduced. Even the initial response to treatment in AL-amyloidosis is limited with one study showing only a 20% response. Restoration of renal function can be achieved by renal transplantation. In one series, 65% of transplanted patients with amyloidosis (15 of them with AL type) were alive 5 years after transplantation. Systemic complications are responsible for the majority of the post-transplant morbidity and mortality in these patients. Unfortunately, for most patients with this condition the overall prognosis is poor and life span limited. Although long survival has been reported occasionally in AL-amyloidosis, amyloid deposition is usually relentless, irreversible and fatal 2-3 years after diagnosis. Since currently these cases are being diagnosed earlier, there is hope that this bleak prognosis may change in the not too distant future. The fact that the primary sequences of some of the amyloidogenic light chains has been determined will contribute to the development of pharmacologic compounds that can inhibit or prevent renal damage in AL-amyloidosis. There are also in vitro and in vivo studies in progress aiming at determining the mechanisms responsible for the localization of amyloid deposits to particular organs and identifying cellular or humoral factors that can accelerate or slow down amyloid deposition. We are currently working with an animal model of AL-amyloidogenesis which will hopefully allow testing of various therapeutic maneuvers to control, ameliorate or reverse renal amyloidosis. References: Bellotti, V., Mangione, P., Merlini, Giampaolo. Immunoglobulin light chain amyloidosis-the archetype of structural and pathogenic variability. J Struct Bio 130:280-289, 2000. 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