http://informahealthcare.com/amy ISSN: 1350-6129 (print), 1744-2818 (electronic) Amyloid, 2014; 21(1): 57–61 ! 2014 Informa UK Ltd. DOI: 10.3109/13506129.2013.851076
CASE REPORT
Systemic AL amyloidosis with unusual cutaneous presentation unmasked by carotenoderma Helena Hu˚lkova´1*, Jan Svojanovsky´2*, Kamil Sˇevela2, Darja Krusova´2, Josef Hanusˇ2, Petr Veˇzda3, Miroslav Soucˇek2, Ivana Ma´rova´4, Josef Feit5, Iva Zambo6, Milica Kovacˇevicova7y, Hana Vla´sˇkova´1, Veronika Kostrouchova´8, Petr Nova´k9, Zdenek Kostrouch8, and Milan Elleder1z 1
Institute of Inherited Metabolic Disorders, Charles University in Prague, First Faculty of Medicine, General University Hospital, Prague, Czech Republic, 2Second Internal Clinic, Masaryk University in Brno, Faculty of Medicine, St. Anne’s University Hospital, Brno, Czech Republic, 3INNEF a.s., Brno, Czech Republic, 4Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic, 5Institute of Pathology, Masaryk University Brno, Faculty of Medicine and University Hospital Brno, Czech Republic, 6First Institute of Pathologic Anatomy, Masaryk University in Brno, Faculty of Medicine and St. Anne’s University Hospital, Brno, Czech Republic, 7Department of Dermatovenerology, Faculty of Medicine, Masaryk University in Brno, Brno, Czech Republic, 8Laboratory of Molecular Pathology, Institute of Cellular Biology and Pathology, Charles University in Prague, First Faculty of Medicine, Prague, Czech Republic, and 9Laboratory of Molecular Structure Characterisation, Institute of Microbiology, Czech Academy of Sciences, Videnska, Prague, Czech Republic Abstract
Keywords
We present a case study of an elderly woman with systemic lambda-type AL amyloidosis that featured unusually extensive cutaneous involvement. The case initially presented with a sudden hyper b-carotenemia with carotenoderma that instigated the clinical examination including skin biopsy. A diagnosis of systemic amyloidosis was made. Immunohistochemistry and Western-blot analysis indicated the presence of lambda light chain proteins in skin amyloid deposits. However, notable co-deposition of wild-type apoA-I and transthyretin was observed which caused initial diagnostic confusion. Proteomic analysis of microdissected skin amyloid deposits by mass spectrometry confirmed lambda light chain proteins in amyloid deposits and co-deposition of apolipoprotein A-IV and serum amyloid P-component. The patient died from renal failure caused by amyloid nephropathy combined with analgesic nephropathy. The autopsy disclosed vascular, cardiac, renal and pulmonary amyloid deposition. While all amyloid deposits were positive for lambda light chain proteins, the immunodetection of apoA-I and transthyretin varied significantly among the visceral amyloid deposits. Although the patient exhibited a 1000-fold increase in serum b-carotene levels, only a mild increase in retinol and lutein concentrations was observed. Increased b-carotene values were also found in the liver and the skin. The mechanisms underlying this hyper b-carotenemia remain undetermined.
Diffuse cutaneous involvement, hypercarotenemia, immunoglobulin lightchain amyloid lambda type History Received 10 July 2013 Revised 7 August 2013 Accepted 30 September 2013 Published online 15 November 2013
Introduction b-fibril aggregation is one of the various types of immunoglobulin light-chain pathologies [1]. Several of the mechanisms and determinants that promote the fibrillar aggregation of light-chain produced by pathologic B cell clones and the
*These authors contributed equally to this work. y Present address: Milica Kovacˇevicˇova´, Landeskrankenhaus Feldkirch, Carinagasse 47, 6800 Feldkirch, Austria z Deceased. Address for correspondence: Helena Hu˚lkova´, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Ke Karlovu 2, 12000 Prague 2, Czech Republic. E-mail: [email protected] Zdenek Kostrouch, Institute of Celluar Biology and Pathology, Charles University in Prague, First Faculty of Medicine, Ke Karlovu 2, 120 00 Prague 2, Czech Republic. E-mail: [email protected]
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Abbreviations: AL: immunoglobulin light-chain amyloid; CKD: chronic kidney disease; GFR: glomerular filtration rate; HD: haemodialysis; PTFE: polytetrafluorethylene
factors that are responsible for the pattern of amyloid deposition at the organ level have been identified [2]. We present a case of systemic lambda-type AL amyloidosis with unusually extensive cutaneous amyloid deposition that was combined with carotenoderma linked with a 1000fold increase in serum b-carotene concentrations. Initial diagnostic attempts were hindered by the simultaneous deposition of two potentially amyloidogenic proteins in the skin and the lack of a sensitive and specific diagnostic antilambda antibody. Mass spectrometric analysis showed predominant presence of lambda light chain proteins in microdissected skin amyloid deposits and the co-deposition of apolipoprotein A-IV and amyloid P-component. The AL nature was finally confirmed by a highly specific and sensitive anti lambda light-chain antibody in the skin as
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well as visceral amyloid deposites that showed variable and often absent co-deposition of other potential amyloidogenic proteins.
Clinical history A female patient, born in 1937, was initially examined in the Nephrological Outpatient Department in May 2004. Case history analysis revealed that the patient had been hypertensive for 10 years, had received arthralgia treatment for 15 years and had a history of recurrent cephalalgia. The patient used analgesics for both arthralgia and cephalalgia for many years. In 2002, renal insufficiency was recorded. The patient had serum creatinine levels of 212 mmol/l and estimated glomerular filtration rate (GFR) of 29.28 ml/min. This was classified as chronic kidney disease (CKD) stage 3– 4 and uremic syndrome with microhaematuria and proteinuria (1.05 g/day). The patient was diagnosed with chronic tubulointerstitial nephritis, which was tentatively classified as analgesic nephropathy. In 2005 and 2006, the patient stabilized; estimated GFR dropped slightly to values consistent with stage 4 CKD, and proteinuria was reduced to 0.5 g/ day. In 2007, the patient was admitted to the hospital for sudden orange skin colouration accompanied by pruritus. A skin biopsy disclosed amyloidosis. Outpatient examinations performed in 2007 and 2008 revealed the gradual incremental progression of chronic renal insufficiency to CKD stage 5. The patient began receiving a regular haemodialysis programme in March 2009. In November 2009, the patient’s health deteriorated. During the hospitalization in December 2009, the neurologist diagnosed extrapyramidal syndrome and organic psycho-syndrome. The obvious orange colour of the patient’s trunk and proximal areas of the limbs and face persisted, namely periorbitally. Scar tissue, that formed in the skin after probationary excisions, was normal in colour. In spite of complex treatment, the patient’s state continued to deteriorate and progressed to unconsciousness. The patient died after 59 days of hospitalization.
Materials and methods Methods of amyloid analysis Classical histology and electron microscope analysis of amyloid deposits were performed using standard techniques for amyloid detection. The antibodies used were: anti-serum amyloid P-component, anti-apolipoprotein A-I, anti-apolipoprotein E, anti-b2 microglobulin (all Abcam, Cambridge, UK), anti-serum amyloid A protein, anti-transthyretin, antiimmunoglobulin light-chain kappa, anti-immunoglobulin light-chain lambda, anti-chymotrypsin, anti-lysozyme and anti-fibrinogen (Dako, Glostrup, Denmark). A highly specific and sensitive mouse monoclonal antibody against immunoglobulin light-chain lambda (pwlam) was kindly provided by Prof. P. Westermark (Uppsala, Sweden). Details and a list of the specific immunohistochemical methods are given in Supplementary Table 1. SDS–PAGE and Western blotting were perfomed as previously described [3]. Mass spectrometry analysis was performed as previously described [4], with modifications.
Amyloid, 2014; 21(1): 57–61
Examination of serum and tissue carotenoids was perfomed as described [5]. For detailed methods and the list of biochemical and histopathological examinations, please see Supplementary Information.
Results Skin biopsy Amyloid deposits displayed Congo red-induced dichroism and a strong signal characteristic of the serum amyloid P-component present as a continuous, homogenous subepidermal layer that completely obscured the papillary and reticular layers (Figure 1A). Fibrillin, which is normally present throughout the skin, was completely absent from the amyloid deposits and only detectable in the dermis (Figure 1B). Periadnexal amyloid deposition was also observed around vessels and nerves and deep in the dermis around elastic fibres. In situ immunohistochemical analysis of amyloid deposits showed biophysical features typical of amyloid b fibrils, including Congo red dichroism, serum amyloid P-component and thioflavin T affinity; the ultrastructure was characteristic of unbranched microfibrils. Immunoglobulin light-chain kappa immunohistochemistry yielded an irregular signal. The signals for apoA-I and transthyretin were strong and uniform (Figure 1C and D, respectively). This led to suspicion of genetic amyloidosis on the basis of apoA-I or transthyretin. However, DNA sequencing showed that both of these genes had wild-type sequences (data not shown). The immunoprofile of the amyloid deposits is summarized in Supplementary Table 1. Analysis of the amyloidogenic proteins extracted from the skin biopsy sample by Western blots revealed elevated levels of dimeric Ig light-chain lambda molecules (50 kDa), transthyretin and apoA-I compared to a control age- and location-matched skin tissue sample. Monomeric light-chain lambda proteins (25 kDa) were visible only at longer exposures and were weaker compared to the control sample. The detection of lambda chain proteins (by the commercial lightchain lambda antibody) was weaker in comparison to kappa proteins and transthyretin extractable from the control tissue (Supplementary Figure 1). Amyloid deposits in skin cryosections were microdissected and analyzed by mass spectrometry. This approach revealed a predominant presence of Ig light-chain lambda proteins and co-deposition of apolipoprotein A-IV and serum amyloid P-component proteins in amyloid deposits. A list of the proteins found in the amyloid deposits by mass spectrometry is given in Supplementary Table 2. Biochemical analysis of serum samples conducted during hospitalization (after December 2009) showed elevated levels of free lambda light-chains (792.50 mg/l; normal range 5.70–26.30), normal, although higher levels of kappa light-chains (18.10 mg/l; normal range 3.30–19.40) and the free light-chain kappa/lambda ratio of 0.02 (normal range 0.26–1.65). This supported a diagnosis of light-chain plasma cell dyscrasia. The serum analysis showed substantially increased levels of b-carotene, up to 1000-fold greater than normal levels, which was keeping with carotenoderma (Supplementary Table 3A) with the deposition of b-carotene
DOI: 10.3109/13506129.2013.851076
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Figure 1. Histological analysis of amyloid deposits. (A–E) Skin bioptic samples. (A) A continuous band of amyloid stained strongly for the serum amyloid P component in the skin (arrows). The dermal staining is given partly by amyloid, partly by normal dermal extracellular matrix (obj. 20). (B) Staining for fibrillin indicates its absence in the amyloid band (arrows). Its staining in the dermal area is physiological (obj. 10). (C) Strong staining of the amyloid band for apoA-I (arrows) (obj. 20). (D) Strong staining of the amyloid band for transthyretin (arrows) (obj. 20). (E–G) Detection of immunoglobulin lambda proteins using a highly specific antibody (gift). (E) Strong staining of the skin amyloid deposits for immunoglobulin LC lambda proteins (arrows) (obj. 20). (F) Strong staining of the discrete amyloid deposits in the heart (arrows) (obj. 10). (G) Strong staining around the pulmonary alveoli (arrows) (obj. 40). (H) Electron microscopy. Electron microscopy shows amyloid fibrilar network on the surface (labelled by arrowheads) of the pneumonocytes communicating thus freely with the alveloar space (magnification 15 000).
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in the keratin layer of the epidermis (Supplementary Figure 2). Serum levels of retinol and lutein were doubled, while the serum level of lycopene was normal. Amyloid distribution revealed in autopsy. Authopsy revealed amyloid depositions in skin and in vascular walls in most organs. Focal amyloid deposits were found in the thyroid gland, in the intestine, around some glomeruli, throughout the kidney interstitium, in the tongue muscle, in the perineurium and along the basement membranes of the salivary glands. Immunohistochemical examination showed consistent strong labelling of all amyloid deposits by the highly specific immunoglobulin light-chain lambda antibody pwlam that were found in the skin (Figure 1E), in the heart, where amyloid was present in the form of thin layers around the heart trabeculae (Figure 1F), and in the intramural arteries. In the lungs, amyloid deposition occurred in a discrete, uniform layer on the alveolar surface (Figure 1G). Contrary to the labelling for lambda light-chain proteins, the immunohistochemical detection of apoA-I, apoE and kappa light-chain proteins varied between deposits and often were weak or undetectable (Supplementary Table 1). Electron microscopy confirmed the presence of amyloid fibrils on the surface of pneumonocytes (Figure 1H) and the discrete deposition of amyloid inside alveolar septa. A search for a chronic inflammatory process detected mild to more severe chronic inflammation in areas surrounding the renal pelvis. The only signs of b-carotene deposition due to hyper b-carotenemia were observed in the corneal layer of the epidermis. Ito cells were present and contained the usual discrete lipid vacuoles. The concentrations of b-carotene and other detected carotenoids in the liver and skin are listed in Supplementary Table 3B.
Discussion In this particular case, the diagnosis evolved over time. Initially, the strong signals of both apoA-I and transthyretin in the cutaneous amyloid biopsy specimen made it difficult to identify the pathogenic protein. Both proteins are known to be amyloidogenic and co-deposition has been observed during amyloidogenesis [6,7]. Moreover, both apoA-I and transthyretin have been described to cause cutaneous manifestations in otherwise systemic amyloidoses [8]. The co-deposition of multiple amyloidogenic proteins in amyloid foci, as has been described here and elsewhere, can seriously complicate the diagnostic process. This may be aggravated by the uneven sensitivity of antibodies used for the detection of amyloidogenic proteins. apoE is known to participate in the majority, if not in all types, of amyloidoses [9]. In our case, a diagnosis of AL amyloidosis was not obtained initially due to inconclusive results with the commercially available antibodies against Ig light-chains; additionally, serum lambda chain protein levels were not initially specified. Moreover, mild serum light-chain levels can be physiological and should not be misinterpreted as an indication of AL amyloidosis [10]. Microdissection and mass spectrometric analysis of skin amyloid deposits is a direct and unbiased technique for protein analysis that also provides quantitative data. In our case, this
Amyloid, 2014; 21(1): 57–61
technique established the predominant composition of amyloid deposits from Ig lambda chains together with serum amyloid Pcomponent and apoA-IV proteins. The diagnosis of AL amyloidosis was finally supported by the use of a sensitive and specific monoclonal antibody against lambda chain, which produced strong, uniform staining of all amyloid deposits irrespective of their size or organ localization (Supplementary Table 1). This corresponded to a significant increase in serum concentration of free lambda chain proteins. However, a pathologic plasma cell clone was not identified by histological examination in the bone marrow. This case was unusual for the extensive cutaneous amyloid deposition combined with carotenoderma. The latter developed suddenly and initiated the diagnostic process. The diffuse pattern of skin discolouration differed from the classical cutaneous manifestation of hypercarotenemia, which is restricted to predisposed regions. Nevertheless, even systemic discolouration has been described, albeit rarely [11]. The high concentration of b-carotene cannot be explained at this time. Normally, b-carotene is converted to retinol by liver carotene dioxygenase. The increased levels of retinol in the reported case demonstrate significant liver carotene dioxygenase activity and exclude a deficiency of this enzyme as the cause of b-carotene accumulation. The extensive, diffuse pattern of cutaneous amyloid deposition was unusual and the nature of the factors responsible for cutaneous amyloid deposition in general remains unknown. In contrast to extensive cutaneous amyloidoses based on cytokeratin [12] systemic cutaneous amyloidosis is a rare condition and may be part of systemic amyloidosis [13]. Although a comparable case of diffuse, clinically skinlimited amyloidosis has been previously described, it was not investigated further [14]. Our case raises the question of whether predisposition for cutaneous amyloid deposition can be correlated with hypercarotenemia. However, we consider the association between cutaneous amyloid deposition and hypercarotenemia to be coincidental because the carotene-based orange colour did not match with areas of amyloid deposition. The presence of amyloid in normal (i.e. not discoloured) skin areas (not shown) argues against b-carotene as the primary amyloidogenic factor. Currently, no evidence exists supporting the interference of b-carotenes with amyloid deposition. This would also contradict the hypothesis that increased levels of antioxidants, including b-carotenes, inhibit amyloidogenesis and plaque formation to exert amyloid fibril destabilizing effects in Alzheimer’s disease [15]. The unexpected features exhibited by the presented case are further discussed in Supplementary Discussion.
Acknowledgements The authors thank Dr. Helena Brˇ´ızova´ from the Department of Pathology, 2nd Faculty of Medicine, Charles University in Prague for her kind help with the amyloid deposit microdissection. Authors thank Dr. Marke´ta Kostrouchova´ for critical reading of the manuscript.
Declaration of interest The authors declare no conflicts of interest. The study and the authors (indicated in brackets) were supported by the
DOI: 10.3109/13506129.2013.851076
following grants: MSM 0021620806 (H.H., M.E., J.L., H.V. and Z.K.); from the Ministry of Education, Youth and Sports, Czech Republic; MUNI/A/1012/2009 (Z.I.) from Masaryk University in Brno; CZ.1.05/2.1.00/01.0012 ‘‘Centre for Materials Research at FCH BUT’’ from ERDF (IM); UNCE 204022 (Z.K.); PRVOUK-P27/LF1/1 (V.K. and Z.K.), UNCE 204011 and PRVOUK-P24/LF1/3 (H.H. and H.V.) from Charles University in Prague and RVO-VFN64165/2012 (H.V.) from the Ministry of Health of the Czech Republic; Institutional Research Concept RVO 61388971 (P.N.), Centre of microbiology and immunology (CZ.1.07/2.3.00/20.0055 and CZ.1.07/2.3.00/30.0003), ‘‘BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec’’ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund (V.K., P.N. and Z.K.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
References 1. Stevens FJ, Argon Y. Pathogenic light chains and the B-cell repertoire. Immunol Today 1999;20:451–7. 2. Comenzo RL, Wally J, Kica G, Murray J, Ericsson T, Skinner M, Zhang Y. Clonal immunoglobulin light chain variable region germline gene use in AL amyloidosis: association with dominant amyloid-related organ involvement and survival after stem cell transplantation. Br J Haematol 1999;106:744–51. 3. Liby P, Kostrouchova M, Pohludka M, Yilma P, Hrabal P, Sikora J, Brozova E, et al. Elevated and deregulated expression of HDAC3 in human astrocytic glial tumours. Folia Biol (Praha) 2006;52: 21–33.
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4. Stodulkova E, Novak P, Deininger SO, Man P, Capkova J, Kavan D, Ivaskova E, et al. LC MALDI-TOF MS/MS and LC ESI FTMS analyses of HLA-B27 associated peptides isolated from peripheral blood cells. Immunol Lett 2008;116:79–85. 5. Kankova K, Marova I, Zahejsky J, Muzik J, Stejskalova A, Znojil V, Vacha J. Polymorphisms 1704G/T and 2184A/G in the RAGE gene are associated with antioxidant status. Metabolism 2001;50: 1152–60. 6. Buxbaum JN. The systemic amyloidoses. Curr Opin Rheumatol 2004;16:67–75. 7. Sakata N, Hoshii Y, Nakamura T, Kiyama M, Arai H, Omoto M, Morimatsu M, et al. Colocalization of apolipoprotein AI in various kinds of systemic amyloidosis. J Histochem Cytochem 2005;53: 237–42. 8. Rocha N, Velho G, Horta M, Martins A, Massa A. Cutaneous manifestations of familial amyloidotic polyneuropathy. J Eur Acad Dermatol Venereol 2005;19:605–7. 9. Sadowski MJ, Pankiewicz J, Scholtzova H, Mehta PD, Prelli F, Quartermain D, Wisniewski T. Blocking the apolipoprotein E/ amyloid-beta interaction as a potential therapeutic approach for Alzheimer’s disease. Proc Natl Acad Sci USA 2006;103:18787–92. 10. Lachmann HJ, Booth DR, Booth SE, Bybee A, Gilbertson JA, Gillmore JD, Pepys MB, et al. Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis. N Engl J Med 2002; 346:1786–91. 11. Haught JM, Patel S, English III JC. Xanthoderma: a clinical review. J Am Acad Dermatol 2007;57:1051–8. 12. Bourke JF, Berth-Jones J, Burns DA. Diffuse primary cutaneous amyloidosis. Br J Dermatol 1992;127:641–4. 13. Hamidi Asl L, Liepnieks JJ, Hamidi Asl K, Uemichi T, Moulin G, Desjoyaux E, Loire R, et al. Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1. Am J Pathol 1999;154: 221–7. 14. Hung CC, Wang CM, Hong HS, Kuo TT. Unusual skin manifestation of cutaneous amyloidosis. Dermatology 2003;207:65–7. 15. Ono K, Hamaguchi T, Naiki H, Yamada M. Anti-amyloidogenic effects of antioxidants: implications for the prevention and therapeutics of Alzheimer’s disease. Biochim Biophys Acta 2006;1762: 575–86.
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CASE REPORT
Systemic AL amyloidosis with unusual cutaneous presentation unmasked by carotenoderma Helena Hu˚lkova´1*, Jan Svojanovsky´2*, Kamil Sˇevela2, Darja Krusova´2, Josef Hanusˇ2, Petr Veˇzda3, Miroslav Soucˇek2, Ivana Ma´rova´4, Josef Feit5, Iva Zambo6, Milica Kovacˇevicova7y, Hana Vla´sˇkova´1, Veronika Kostrouchova´8, Petr Nova´k9, Zdenek Kostrouch8, and Milan Elleder1z 1
Institute of Inherited Metabolic Disorders, Charles University in Prague, First Faculty of Medicine, General University Hospital, Prague, Czech Republic, 2Second Internal Clinic, Masaryk University in Brno, Faculty of Medicine, St. Anne’s University Hospital, Brno, Czech Republic, 3INNEF a.s., Brno, Czech Republic, 4Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic, 5Institute of Pathology, Masaryk University Brno, Faculty of Medicine and University Hospital Brno, Czech Republic, 6First Institute of Pathologic Anatomy, Masaryk University in Brno, Faculty of Medicine and St. Anne’s University Hospital, Brno, Czech Republic, 7Department of Dermatovenerology, Faculty of Medicine, Masaryk University in Brno, Brno, Czech Republic, 8Laboratory of Molecular Pathology, Institute of Cellular Biology and Pathology, Charles University in Prague, First Faculty of Medicine, Prague, Czech Republic, and 9Laboratory of Molecular Structure Characterisation, Institute of Microbiology, Czech Academy of Sciences, Videnska, Prague, Czech Republic Abstract
Keywords
We present a case study of an elderly woman with systemic lambda-type AL amyloidosis that featured unusually extensive cutaneous involvement. The case initially presented with a sudden hyper b-carotenemia with carotenoderma that instigated the clinical examination including skin biopsy. A diagnosis of systemic amyloidosis was made. Immunohistochemistry and Western-blot analysis indicated the presence of lambda light chain proteins in skin amyloid deposits. However, notable co-deposition of wild-type apoA-I and transthyretin was observed which caused initial diagnostic confusion. Proteomic analysis of microdissected skin amyloid deposits by mass spectrometry confirmed lambda light chain proteins in amyloid deposits and co-deposition of apolipoprotein A-IV and serum amyloid P-component. The patient died from renal failure caused by amyloid nephropathy combined with analgesic nephropathy. The autopsy disclosed vascular, cardiac, renal and pulmonary amyloid deposition. While all amyloid deposits were positive for lambda light chain proteins, the immunodetection of apoA-I and transthyretin varied significantly among the visceral amyloid deposits. Although the patient exhibited a 1000-fold increase in serum b-carotene levels, only a mild increase in retinol and lutein concentrations was observed. Increased b-carotene values were also found in the liver and the skin. The mechanisms underlying this hyper b-carotenemia remain undetermined.
Diffuse cutaneous involvement, hypercarotenemia, immunoglobulin lightchain amyloid lambda type History Received 10 July 2013 Revised 7 August 2013 Accepted 30 September 2013 Published online 15 November 2013
Introduction b-fibril aggregation is one of the various types of immunoglobulin light-chain pathologies [1]. Several of the mechanisms and determinants that promote the fibrillar aggregation of light-chain produced by pathologic B cell clones and the
*These authors contributed equally to this work. y Present address: Milica Kovacˇevicˇova´, Landeskrankenhaus Feldkirch, Carinagasse 47, 6800 Feldkirch, Austria z Deceased. Address for correspondence: Helena Hu˚lkova´, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Ke Karlovu 2, 12000 Prague 2, Czech Republic. E-mail: [email protected] Zdenek Kostrouch, Institute of Celluar Biology and Pathology, Charles University in Prague, First Faculty of Medicine, Ke Karlovu 2, 120 00 Prague 2, Czech Republic. E-mail: [email protected]
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Abbreviations: AL: immunoglobulin light-chain amyloid; CKD: chronic kidney disease; GFR: glomerular filtration rate; HD: haemodialysis; PTFE: polytetrafluorethylene
factors that are responsible for the pattern of amyloid deposition at the organ level have been identified [2]. We present a case of systemic lambda-type AL amyloidosis with unusually extensive cutaneous amyloid deposition that was combined with carotenoderma linked with a 1000fold increase in serum b-carotene concentrations. Initial diagnostic attempts were hindered by the simultaneous deposition of two potentially amyloidogenic proteins in the skin and the lack of a sensitive and specific diagnostic antilambda antibody. Mass spectrometric analysis showed predominant presence of lambda light chain proteins in microdissected skin amyloid deposits and the co-deposition of apolipoprotein A-IV and amyloid P-component. The AL nature was finally confirmed by a highly specific and sensitive anti lambda light-chain antibody in the skin as
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well as visceral amyloid deposites that showed variable and often absent co-deposition of other potential amyloidogenic proteins.
Clinical history A female patient, born in 1937, was initially examined in the Nephrological Outpatient Department in May 2004. Case history analysis revealed that the patient had been hypertensive for 10 years, had received arthralgia treatment for 15 years and had a history of recurrent cephalalgia. The patient used analgesics for both arthralgia and cephalalgia for many years. In 2002, renal insufficiency was recorded. The patient had serum creatinine levels of 212 mmol/l and estimated glomerular filtration rate (GFR) of 29.28 ml/min. This was classified as chronic kidney disease (CKD) stage 3– 4 and uremic syndrome with microhaematuria and proteinuria (1.05 g/day). The patient was diagnosed with chronic tubulointerstitial nephritis, which was tentatively classified as analgesic nephropathy. In 2005 and 2006, the patient stabilized; estimated GFR dropped slightly to values consistent with stage 4 CKD, and proteinuria was reduced to 0.5 g/ day. In 2007, the patient was admitted to the hospital for sudden orange skin colouration accompanied by pruritus. A skin biopsy disclosed amyloidosis. Outpatient examinations performed in 2007 and 2008 revealed the gradual incremental progression of chronic renal insufficiency to CKD stage 5. The patient began receiving a regular haemodialysis programme in March 2009. In November 2009, the patient’s health deteriorated. During the hospitalization in December 2009, the neurologist diagnosed extrapyramidal syndrome and organic psycho-syndrome. The obvious orange colour of the patient’s trunk and proximal areas of the limbs and face persisted, namely periorbitally. Scar tissue, that formed in the skin after probationary excisions, was normal in colour. In spite of complex treatment, the patient’s state continued to deteriorate and progressed to unconsciousness. The patient died after 59 days of hospitalization.
Materials and methods Methods of amyloid analysis Classical histology and electron microscope analysis of amyloid deposits were performed using standard techniques for amyloid detection. The antibodies used were: anti-serum amyloid P-component, anti-apolipoprotein A-I, anti-apolipoprotein E, anti-b2 microglobulin (all Abcam, Cambridge, UK), anti-serum amyloid A protein, anti-transthyretin, antiimmunoglobulin light-chain kappa, anti-immunoglobulin light-chain lambda, anti-chymotrypsin, anti-lysozyme and anti-fibrinogen (Dako, Glostrup, Denmark). A highly specific and sensitive mouse monoclonal antibody against immunoglobulin light-chain lambda (pwlam) was kindly provided by Prof. P. Westermark (Uppsala, Sweden). Details and a list of the specific immunohistochemical methods are given in Supplementary Table 1. SDS–PAGE and Western blotting were perfomed as previously described [3]. Mass spectrometry analysis was performed as previously described [4], with modifications.
Amyloid, 2014; 21(1): 57–61
Examination of serum and tissue carotenoids was perfomed as described [5]. For detailed methods and the list of biochemical and histopathological examinations, please see Supplementary Information.
Results Skin biopsy Amyloid deposits displayed Congo red-induced dichroism and a strong signal characteristic of the serum amyloid P-component present as a continuous, homogenous subepidermal layer that completely obscured the papillary and reticular layers (Figure 1A). Fibrillin, which is normally present throughout the skin, was completely absent from the amyloid deposits and only detectable in the dermis (Figure 1B). Periadnexal amyloid deposition was also observed around vessels and nerves and deep in the dermis around elastic fibres. In situ immunohistochemical analysis of amyloid deposits showed biophysical features typical of amyloid b fibrils, including Congo red dichroism, serum amyloid P-component and thioflavin T affinity; the ultrastructure was characteristic of unbranched microfibrils. Immunoglobulin light-chain kappa immunohistochemistry yielded an irregular signal. The signals for apoA-I and transthyretin were strong and uniform (Figure 1C and D, respectively). This led to suspicion of genetic amyloidosis on the basis of apoA-I or transthyretin. However, DNA sequencing showed that both of these genes had wild-type sequences (data not shown). The immunoprofile of the amyloid deposits is summarized in Supplementary Table 1. Analysis of the amyloidogenic proteins extracted from the skin biopsy sample by Western blots revealed elevated levels of dimeric Ig light-chain lambda molecules (50 kDa), transthyretin and apoA-I compared to a control age- and location-matched skin tissue sample. Monomeric light-chain lambda proteins (25 kDa) were visible only at longer exposures and were weaker compared to the control sample. The detection of lambda chain proteins (by the commercial lightchain lambda antibody) was weaker in comparison to kappa proteins and transthyretin extractable from the control tissue (Supplementary Figure 1). Amyloid deposits in skin cryosections were microdissected and analyzed by mass spectrometry. This approach revealed a predominant presence of Ig light-chain lambda proteins and co-deposition of apolipoprotein A-IV and serum amyloid P-component proteins in amyloid deposits. A list of the proteins found in the amyloid deposits by mass spectrometry is given in Supplementary Table 2. Biochemical analysis of serum samples conducted during hospitalization (after December 2009) showed elevated levels of free lambda light-chains (792.50 mg/l; normal range 5.70–26.30), normal, although higher levels of kappa light-chains (18.10 mg/l; normal range 3.30–19.40) and the free light-chain kappa/lambda ratio of 0.02 (normal range 0.26–1.65). This supported a diagnosis of light-chain plasma cell dyscrasia. The serum analysis showed substantially increased levels of b-carotene, up to 1000-fold greater than normal levels, which was keeping with carotenoderma (Supplementary Table 3A) with the deposition of b-carotene
DOI: 10.3109/13506129.2013.851076
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Figure 1. Histological analysis of amyloid deposits. (A–E) Skin bioptic samples. (A) A continuous band of amyloid stained strongly for the serum amyloid P component in the skin (arrows). The dermal staining is given partly by amyloid, partly by normal dermal extracellular matrix (obj. 20). (B) Staining for fibrillin indicates its absence in the amyloid band (arrows). Its staining in the dermal area is physiological (obj. 10). (C) Strong staining of the amyloid band for apoA-I (arrows) (obj. 20). (D) Strong staining of the amyloid band for transthyretin (arrows) (obj. 20). (E–G) Detection of immunoglobulin lambda proteins using a highly specific antibody (gift). (E) Strong staining of the skin amyloid deposits for immunoglobulin LC lambda proteins (arrows) (obj. 20). (F) Strong staining of the discrete amyloid deposits in the heart (arrows) (obj. 10). (G) Strong staining around the pulmonary alveoli (arrows) (obj. 40). (H) Electron microscopy. Electron microscopy shows amyloid fibrilar network on the surface (labelled by arrowheads) of the pneumonocytes communicating thus freely with the alveloar space (magnification 15 000).
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in the keratin layer of the epidermis (Supplementary Figure 2). Serum levels of retinol and lutein were doubled, while the serum level of lycopene was normal. Amyloid distribution revealed in autopsy. Authopsy revealed amyloid depositions in skin and in vascular walls in most organs. Focal amyloid deposits were found in the thyroid gland, in the intestine, around some glomeruli, throughout the kidney interstitium, in the tongue muscle, in the perineurium and along the basement membranes of the salivary glands. Immunohistochemical examination showed consistent strong labelling of all amyloid deposits by the highly specific immunoglobulin light-chain lambda antibody pwlam that were found in the skin (Figure 1E), in the heart, where amyloid was present in the form of thin layers around the heart trabeculae (Figure 1F), and in the intramural arteries. In the lungs, amyloid deposition occurred in a discrete, uniform layer on the alveolar surface (Figure 1G). Contrary to the labelling for lambda light-chain proteins, the immunohistochemical detection of apoA-I, apoE and kappa light-chain proteins varied between deposits and often were weak or undetectable (Supplementary Table 1). Electron microscopy confirmed the presence of amyloid fibrils on the surface of pneumonocytes (Figure 1H) and the discrete deposition of amyloid inside alveolar septa. A search for a chronic inflammatory process detected mild to more severe chronic inflammation in areas surrounding the renal pelvis. The only signs of b-carotene deposition due to hyper b-carotenemia were observed in the corneal layer of the epidermis. Ito cells were present and contained the usual discrete lipid vacuoles. The concentrations of b-carotene and other detected carotenoids in the liver and skin are listed in Supplementary Table 3B.
Discussion In this particular case, the diagnosis evolved over time. Initially, the strong signals of both apoA-I and transthyretin in the cutaneous amyloid biopsy specimen made it difficult to identify the pathogenic protein. Both proteins are known to be amyloidogenic and co-deposition has been observed during amyloidogenesis [6,7]. Moreover, both apoA-I and transthyretin have been described to cause cutaneous manifestations in otherwise systemic amyloidoses [8]. The co-deposition of multiple amyloidogenic proteins in amyloid foci, as has been described here and elsewhere, can seriously complicate the diagnostic process. This may be aggravated by the uneven sensitivity of antibodies used for the detection of amyloidogenic proteins. apoE is known to participate in the majority, if not in all types, of amyloidoses [9]. In our case, a diagnosis of AL amyloidosis was not obtained initially due to inconclusive results with the commercially available antibodies against Ig light-chains; additionally, serum lambda chain protein levels were not initially specified. Moreover, mild serum light-chain levels can be physiological and should not be misinterpreted as an indication of AL amyloidosis [10]. Microdissection and mass spectrometric analysis of skin amyloid deposits is a direct and unbiased technique for protein analysis that also provides quantitative data. In our case, this
Amyloid, 2014; 21(1): 57–61
technique established the predominant composition of amyloid deposits from Ig lambda chains together with serum amyloid Pcomponent and apoA-IV proteins. The diagnosis of AL amyloidosis was finally supported by the use of a sensitive and specific monoclonal antibody against lambda chain, which produced strong, uniform staining of all amyloid deposits irrespective of their size or organ localization (Supplementary Table 1). This corresponded to a significant increase in serum concentration of free lambda chain proteins. However, a pathologic plasma cell clone was not identified by histological examination in the bone marrow. This case was unusual for the extensive cutaneous amyloid deposition combined with carotenoderma. The latter developed suddenly and initiated the diagnostic process. The diffuse pattern of skin discolouration differed from the classical cutaneous manifestation of hypercarotenemia, which is restricted to predisposed regions. Nevertheless, even systemic discolouration has been described, albeit rarely [11]. The high concentration of b-carotene cannot be explained at this time. Normally, b-carotene is converted to retinol by liver carotene dioxygenase. The increased levels of retinol in the reported case demonstrate significant liver carotene dioxygenase activity and exclude a deficiency of this enzyme as the cause of b-carotene accumulation. The extensive, diffuse pattern of cutaneous amyloid deposition was unusual and the nature of the factors responsible for cutaneous amyloid deposition in general remains unknown. In contrast to extensive cutaneous amyloidoses based on cytokeratin [12] systemic cutaneous amyloidosis is a rare condition and may be part of systemic amyloidosis [13]. Although a comparable case of diffuse, clinically skinlimited amyloidosis has been previously described, it was not investigated further [14]. Our case raises the question of whether predisposition for cutaneous amyloid deposition can be correlated with hypercarotenemia. However, we consider the association between cutaneous amyloid deposition and hypercarotenemia to be coincidental because the carotene-based orange colour did not match with areas of amyloid deposition. The presence of amyloid in normal (i.e. not discoloured) skin areas (not shown) argues against b-carotene as the primary amyloidogenic factor. Currently, no evidence exists supporting the interference of b-carotenes with amyloid deposition. This would also contradict the hypothesis that increased levels of antioxidants, including b-carotenes, inhibit amyloidogenesis and plaque formation to exert amyloid fibril destabilizing effects in Alzheimer’s disease [15]. The unexpected features exhibited by the presented case are further discussed in Supplementary Discussion.
Acknowledgements The authors thank Dr. Helena Brˇ´ızova´ from the Department of Pathology, 2nd Faculty of Medicine, Charles University in Prague for her kind help with the amyloid deposit microdissection. Authors thank Dr. Marke´ta Kostrouchova´ for critical reading of the manuscript.
Declaration of interest The authors declare no conflicts of interest. The study and the authors (indicated in brackets) were supported by the
DOI: 10.3109/13506129.2013.851076
following grants: MSM 0021620806 (H.H., M.E., J.L., H.V. and Z.K.); from the Ministry of Education, Youth and Sports, Czech Republic; MUNI/A/1012/2009 (Z.I.) from Masaryk University in Brno; CZ.1.05/2.1.00/01.0012 ‘‘Centre for Materials Research at FCH BUT’’ from ERDF (IM); UNCE 204022 (Z.K.); PRVOUK-P27/LF1/1 (V.K. and Z.K.), UNCE 204011 and PRVOUK-P24/LF1/3 (H.H. and H.V.) from Charles University in Prague and RVO-VFN64165/2012 (H.V.) from the Ministry of Health of the Czech Republic; Institutional Research Concept RVO 61388971 (P.N.), Centre of microbiology and immunology (CZ.1.07/2.3.00/20.0055 and CZ.1.07/2.3.00/30.0003), ‘‘BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec’’ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund (V.K., P.N. and Z.K.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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