Human Pathology (2014) xx, xxx–xxx

www.elsevier.com/locate/humpath

Original contribution

Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining☆,☆☆ Paisit Paueksakon MD a,⁎, Agnes B. Fogo MD a , Sanjeev Sethi MD b a

Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232–2561, USA Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA

b

Received 11 October 2013; revised 22 February 2014; accepted 28 February 2014

Keywords: Amyloidosis; LECT2; Immunohistochemical staining; Laser microdissection; Mass spectrometry

Summary We investigated the role of leukocyte chemotactic factor (LECT2) immunohistochemical staining in the diagnosis of type of renal amyloidosis. Fifty renal amyloidosis cases with available paraffin blocks in our 2002 to 2012 renal biopsy files were reviewed. Patients were designated as a defined amyloid, including amyloid light chain (AL) and amyloid-associated amyloid (AA), or a nonAL/non-AA amyloid group. LECT2-specific antibody immunohistochemistry was performed in all 50 cases. Laser microdissection and mass spectrometry (LMD/MS) were performed in 10 cases. Forty-five patients had amyloid classified as either AL (44) or AA (1), and 5 had undetermined amyloid. Three of the five non-AL/non-AA group patient biopsies showed positive LECT2 immunohistochemical staining, and of these, LECT2 was also identified by LMD/MS in 1 patient, fibrinogen-α was identified in 1 patient, and apolipoprotein IV was identified in 1 patient. Two of these non-AL/non-AA patients showed negative LECT2 staining, and LMD/MS showed apolipoprotein IV as a major protein component. Five of the 44 AL amyloid patients showed weakly positive LECT2 staining. However, LECT2 was not identified by LMD/MS in any of these 5 cases. The single patient with AA amyloid was negative for LECT2 by immunohistochemical staining. Among 5 non-AL and non-AA amyloidosis patients in our study, 1 had LECT2, 1 had fibrinogen-α, and 3 had apolipoprotein IV as a major protein component. The data from this study show that weak LECT2 staining should be regarded as indeterminate or a negative result and does not per se allow diagnosis of specific amyloid type. The diagnosis of LECT2 renal amyloidosis may require LMD/MS confirmation. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Amyloidosis is a group of diseases characterized by excessive tissue deposits of proteins in an insoluble β-pleated ☆

Disclosures: None No conflict of interest ⁎ Corresponding author. Paisit Paueksakon, MD, Department of Pathology, Microbiology and Immunology MCN C2318B Vanderbilt University Medical Center 1161 21st Avenue South Nashville, TN 37232–2561, USA. E-mail address: [email protected] (P. Paueksakon). ☆☆

http://dx.doi.org/10.1016/j.humpath.2014.02.020 0046-8177/© 2014 Elsevier Inc. All rights reserved.

sheet format. Amyloid deposits are identified based on their apple-green birefringence under a polarized light on Congo red staining and by the presence of randomly arranged, nonbranching fibrils that measure 7 to 10 nm in diameter on electron microscopy [1]. The subtypes of amyloid are categorized by the chemical composition of the proteins. The most common form of renal amyloidosis is amyloid light chain (AL) or primary amyloidosis, in which fibrils are composed of monoclonal immunoglobulin light chains [2]. Reactive secondary amyloidosis is characterized by tissue deposition of serum amyloid A protein, which is derived from an acute-

2 phase reactant protein synthesized by the liver. Reactive amyloidosis is seen in conditions associated with chronic immune activation including rheumatoid arthritis, ankylosing spondylitis, chronic draining infections (ie, osteomyelitis, chronic skin infections including decubitus ulcers, bronchiectasis), Crohn disease, tuberculosis, and familial Mediterranean fever [2]. Hereditary forms of amyloidosis comprise another group of amyloid that is now being diagnosed with more frequency and include amyloid derived from transthyretins, fibrinogen-α, lysozyme, gelsolin, and apolipoproteins [1,3]. Renal leukocyte chemotactic factor 2 (LECT2) amyloidosis has recently been described [4-6] and shows extensive congophilic deposits in the glomeruli, interstitium, and arteries. LECT2 is a chemotactic factor for neutrophils and has other physiologic roles, including cell growth promotion and repair after damage [5,7,8]. We performed LECT2 immunohistochemical staining on 50 cases of renal amyloidosis defined by Congo red positivity with available paraffin blocks in our laboratory over the past 11 years to assess the diagnostic utility of LECT2 staining in defining amyloid type.

2. Materials and methods Fifty renal amyloidosis cases with available paraffin blocks in our 2002 to 2012 renal biopsy files at Vanderbilt University Medical Center were reviewed. Patients were designated as defined amyloid (AL and AA) or non-AL/nonAA (absence of monoclonal staining pattern for κ and λ by immunofluorescence study and negative amyloid-associated protein immunohistochemistry). To determine the nature of the non-AL/non-AA group and specificity of LECT2 protein staining in diagnosis of amyloidosis type, immunohistochemical study for LECT2-specific antibody was performed in all 50 cases of amyloidosis. Laser microdissection and mass spectrometry (LMD/MS) were performed at Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, in 5 AL patients and 5 non-AL/non-AA patients to determine and confirm subtype of amyloidosis. To demonstrate specificity of LECT2 protein staining, LECT2 immunohistochemistry was also performed on 10 patients with arterionephrosclerosis and 5 patients with diabetic nephropathy with negative Congo red stains and no amyloid fibrils by electron microscopy.

P. Paueksakon et al. with apple-green birefringence under polarizing microscopy. Known amyloid cases served as positive control.

2.2. Immunofluorescence and immunohistochemical studies Samples were placed in Michel’s media, washed in buffer, and frozen in a cryostat. Sections, cut at 2 μm, were rinsed in buffer and reacted with fluorescein-tagged polyclonal rabbit anti-human antibodies to IgG, IgA, IgM, C3, C1q, and κ and λ light chains (all from Dako, Carpenteria, CA) for 1 hour and rinsed, and a coverslip was applied using aqueous mounting media. For AA and LECT2 detection, 2-μm-thick paraffin-embedded sections were cut, deparaffinized, rehydrated, and blocked with normal horse serum for 5 minutes, followed by reaction for 1 hour with a 1:100 dilution of mouse anti-human amyloid A protein monoclonal antibody (mAb) (DakoCytomation, Glostrup, Denmark) or a 1:40 dilution of a goat antihuman LECT2 mAb (Vector Laboratories, Burlingame, CA). Immunoreactions were visualized using 3,3′-diaminobenzidine as the substrate (Dako, Carpenteria, CA). Liver tissue from a patient with cirrhosis was used as a positive control for LECT2 (Fig. 1). Negative controls were done omitting primary antibody.

2.3. Electron microscopy Tissue was allocated to glutaraldehyde, dehydrated using graded alcohols, followed by embedding in Spurr embedding resin. Sections of 1 μm thickness were cut using an ultramicrotome and stained with toluidine blue. Glomeruli were selected, and sections of 70 to 100 nm were cut and examined in a Morgagni Philips FEI transmission electron microscope. Electron photomicrographs were routinely taken at 5600×, 7200×, and 56 000× magnifications, and amyloid fibril diameter was measured.

2.1. Light microscopy Briefly, renal biopsies were fixed in buffered formaldehyde, dehydrated in graded alcohols, and embedded in paraffin using standard techniques. Serial 2- to 3-μm-thick sections were cut and stained with hematoxylin and eosin, Jones methenamine silver, and periodic acid–Schiff reagent. In addition, a 7-μmthick section was cut and stained with Congo red wherein the diagnosis of amyloid was confirmed by Congo red positivity

Fig. 1 Positive control for LECT2 using cirrhotic liver tissue (LECT2 immunostaining; original magnification ×200).

IHC not reliable for LECT2 amyloidosis

3

2.4. Specimen preparation, laser microdissection, and mass spectrometry–based proteomic analysis The method has previously been published [9]. Briefly, thick sections of 10 μm from formalin-fixed, paraffinembedded tissues were stained with Congo red. Glomeruli with positive Congo red areas viewed under fluorescent light source appeared bright red. The Congo red deposits were identified under fluorescent light and microdissected with laser microdissection (LMD). The microdissected material was placed in 0.5 mL microcentrifuge tube caps containing 35 mL Tris/EDTA/0.002% Zwittergent buffer. Microdissected fragments were digested into tryptic peptides overnight and analyzed by liquid chromatography electrospray tandem mass spectrometry (MS). MS raw data files were queried using three different algorithms (Sequest, Mascot, and X!Tandem); the results were combined and assigned peptide and protein probability scores in Scaffold (Proteome Software, Portland, OR). For each case, a list of proteins based on peptides detected by MS was generated. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm. Protein identifications below the 90% confidence level and those with single- peptide identification were not considered in our analysis. The ‘Spectra’ value indicates the total number of mass spectra collected on the mass spectrometer and matched to the protein using the proteomics software. A higher number of mass spectra is indicative of greater abundance and will typically yield greater amino acid sequence coverage. A higher mass spectra value also indicates a higher confidence in the protein identification. Our clinical amyloid testing requires a minimum number of four spectra in all samples before the protein identification is deemed clinically valid. The diagnosis of amyloidosis at the proteomic level using LMD/MS is based on the presence of serum amyloid-P component (SAP) and apolipoprotein E [10,11]. The typing of amyloid is then based on the presence of spectra that correspond to the specific type of amyloid. For instance, AL λ light chain amyloid contains large spectra of Ig λ light chain C-region with or without λ light chain V region; AA amyloidosis shows spectra of SAA protein; LECT2 amyloidosis shows spectra of LECT2 protein; fibrinogen-α amyloidosis shows spectra for fibrinogen-α including the mutated fibrinogen A peptide; transthyretin amyloidosis shows spectra for transthyretin, and so on. LMD/MS of normal glomeruli (day 0 protocol kidney biopsies) showed Table 1

that these glomeruli do not contain spectra of any of these amyloidogenic peptides.

3. Results Among the 10 623 renal biopsy specimens sent to our laboratory over the past 11 years, 50 cases of renal amyloidosis with available paraffin blocks were identified based on applegreen birefringent congophilic deposits that, by electron microscopy, were observed to have typical ultrastructural features of amyloid. Forty-five patients had amyloid classified as either AL (44, 97%) or AA (1, 3%). These patients ranged from 33 to 84 years old (mean 62.1 ± 2.0) (P = .1 vs. non-AL/ non-AA group), male/female ratio 28:17 and white/black ratio 40:5. Five patients had non-AA/non-AL amyloid. These patients were 51 to 74 years old (mean 65.5 ± 5.0), 4 males and 1 female, white/black ratio 3:2 (Table 1). There were no Hispanic patients in this study. Proteinuria was not significantly different between these groups (non-AL/non-AA 3.0 ± 1.0 g/24 h versus 6.4 ± 0.7 in AA/AL, P = .2). There was extensive amyloid deposition with glomerular, interstitial and vascular involvement in all in 5 non-AL nonAA patients (Fig. 2). Three of the five patients in the non-AL/ non-AA group showed positive LECT2 immunohistochemical staining in glomeruli, interstitium, and arteries, and of these, one patient showed strong LECT2 immunostaining (Fig. 3) and 2 patients showed focal, weak LECT2 immunostaining (Fig. 4). These three LECT2 positive patients in the non-AL/ non-AA group were white, non-immigrant residents in the United States. Five of 44 AL amyloid patients showed focal, weakly positive LECT2 immunohistochemical staining in glomeruli and arteries (Table 2). LMD/MS was performed in 10 patients, including 5 patients from the non-AL/non-AA group and 5 patients with AL amyloidosis. LECT2 was also identified by LMD/MS in 1 non-AL/non-AA patient with strong LECT2 immunostaining (Fig. 5), thus leading to a diagnosis of LECT2-associated amyloidosis. In two non-AL/non-AA patients with focal, weak LECT2 immunostaining, LECT2 was not identified by LMD/MS. However, LMD/MS showed an unexpected finding of fibrinogen-α component as a major protein component in one patient, thus leading to a diagnosis of fibrinogen-α type amyloid. In a second patient, apolipoprotein IV was found as a major protein component, thus leading to a diagnosis of apolipoprotein IV amyloid. The remaining 2

Clinical data

Amyloid

N

Age (y)

Male/female

White/black/Hispanic

Uprot (g/24h)

AL AA Non-AL/non-AA

44 1 5

33-84 72 51-74

27/17 1/0 4/1

39/5/0 1/0/0 3/2/0

3.0± 1.0 6.0 6.4± 0.7

Abbreviations: AL, amyloid light chain; AA, amyloid-associated amyloid; Uprot, urine protein.

4

Fig. 2 Extensive mesangial expansion by amorphous eosinophilic acellular material in LECT2-associated renal amyloidosis (Jones' silver stain; original magnification ×400).

non-AL/non-AA patients were negative for LECT2 by immunohistochemical staining and by LMD/MS. LMD/MS showed apolipoprotein IV as a major protein component in both patients, suggesting apolipoprotein IV amyloidosis. In 5 AL patients with focal, weakly positive immunohistochemical staining for LECT2, LECT2 was not identified by LMD/ MS, indicating this was not the cause of amyloid. The single patient with AA amyloid was negative for LECT2 by immunohistochemical staining (Table 3). All patients with diabetic nephropathy and arterionephrosclerosis showed negative LECT2 immunohistochemical staining.

4. Discussion Although immunohistochemistry is the usual method used for typing renal amyloidosis and, in one report, successfully

P. Paueksakon et al.

Fig. 4 Fibrinogen-α amyloidosis with focal, weak LECT2 staining (original magnification ×400).

showed the amyloid type in 96.6% of cases [1], it comes with diagnostic pitfalls, and occasionally cases cannot be definitively typed using this method. Many of these unclassified cases may represent AL, which is occasionally non-reactive with commercial κ or λ light-chain antibodies [12]. It is also conceivable that some of these AL cases not staining for light chains or AA amyloid are due to rare forms of proteins such as LECT2, apolipoproteins, fibrinogen-α, gelsolin, etc, as major protein components. Laser microdissection and mass spectrometry (LMD/MS) have proved to be useful to determine the nature and type of the amyloid protein in cases that could not be typed by our routine immunohistochemical panel [13,14]. The major advantage of LMD/MS over conventional techniques in typing of amyloid is that LMD/MS is a single test that can identify the amyloid protein, in contrast to immunohistochemical studies that may require several antibodies staining multiple sections [15]. Table 2 kidney

Fig. 3 LECT2 immunostaining in a non-AL/non-AA amyloid case in glomeruli and this LECT2 case was confirmed by LMD/MS (original magnification ×200).

Distribution of LECT2-associated amyloid within the

Case Amyloid no. type

Mesangium GBM Interstitium Arteries

1 2 3 4 5 6 7 8 9 10

++++ ++ ++ − − + + + + +

Non-AL/AA Non-AL/AA Non-AL/AA Non-AL/AA Non-AL/AA AL AL AL AL AL

++++ ++ ++ − − + + + + +

++++ ++ ++ − − − − − − −

++++ ++ ++ N/A − + + + + +

Abbreviations: AL, amyloid light chain; AA, amyloid-associated amyloid; GBM, glomerular basement membranes; N/A, arteries not present in the specimen. NOTE. LECT2 deposits: −, absent; +, b25%; ++, 25%-50%; +++, 50%-75%; ++++, 76%-100%.

IHC not reliable for LECT2 amyloidosis

5

Fig. 5 Mass spectrometry by spectra. Representative mass spectrometry data by spectral analyses from a case of LECT2 amyloidosis. The figure shows proteomics data from 3 microdissected samples (sample 1, 2 and 3) from the biopsy showing the presence of apolipoprotein E, serum amyloid-P component, and LECT2. The probability number (N95% is highlighted by green, 80%-94% by yellow) indicates essentially the percent homology between peptides detected in the specimens and the published amino acid sequences of their corresponding proteins.

Moreover, LMD/MS is performed from paraffin-embedded tissue and has no special tissue requirement. However, LMD/ MS is labor intensive and currently not widely available. The common indications for LMD/MS in renal amyloidosis include amyloid type confirmation, insufficient sample for immunofluorescence or immunohistochemical studies, difficult cases on routine renal biopsy studies such as heavy chain amyloidosis, and familial and hereditary amyloidosis [15,16]. The diagnosis of amyloidosis at the proteomic level using LMD/MS is based on the presence of large spectra for proteins that have amyloidogenic properties, in addition to apolipoprotein E and SAP [10]. Therefore, in AL large spectra numbers of light chain constant regions along with apolipoprotein E and SAP are present. In contrast, in LECT2 and fibrinogen-α amyloidosis, large spectra numbers of LECT2 and fibrinogenα chain are present along with apolipoprotein E and SAP (Fig. 4). In our study, LMD/MS identified 3 cases of apolipoprotein A-IV and a single case of fibrinogen-α, which were not identified by conventional immunofluorescence and immunohistochemical studies, and confirmed diagnosis of one case of LECT2 amyloidosis. Table 3

The morphological pattern of amyloid distribution in our case of LECT2 amyloidosis was similar to the previous reports [4-6]; that is, with extensive involvement of all compartments of the kidney. Thus, this pattern is not different from other common types of renal amyloidosis [1]. Our study showed LECT2 immunohistochemical study alone may not be sufficient to make diagnosis of LECT2 amyloidosis in cases with weakly positive staining. The data from this study show that weak LECT2 staining could be considered as a negative result. In other words, establishing a higher threshold for LECT2 immunostaining would have correctly identified all positive and negative LECT2 amyloidosis cases, as defined by LMD/MS results. Little is known about the pathogenesis or prognosis of renal LECT2 amyloidosis. It has been postulated that LECT2 amyloidosis may be a consequence of localized inflammatory process that lead to increased synthesis of the amyloidogenic valine 40–containing LECT2 variant in individuals homozygous for the G-allele [6]. In addition, LECT2 amyloidosis may result from a genetic defect in a protein involved in LECT2 transport [6]. This hypothesis is supported by data

Amyloid classification by IHC and LMD/MS

Amyloid type

Positive LECT2 IHC

LECT2 LMD/MS peak

Other protein components

AL AA Non-AL/non-AA

5 0 3

0 ND 1

None None 2 Apo-IV 1 fibrinogen-α

Abbreviations: AL, amyloid light chain; AA, amyloid-associated amyloid; IHC, immunohistochemistry; ND, not done.

6 from Murphy et al. [6] that showed 7 of 10 renal LECT2 amyloidosis patients were Mexican Americans. Although most patients with LECT2 amyloidosis are Mexican American [6], our patient with LECT2 amyloidosis was white. It is also conceivable that LECT2 is an inducible protein and amyloid precursor which may have a role in fibril formation in cases of apolipoprotein A-IV, fibrinogen-α, and AL amyloidosis. The weak LECT2 staining seen in such cases could support the possibility in our study. In our study, cases of apolipoprotein A-IV and fibrinogen-α amyloid were different from previous reports [1,16]. Thus, apolipoprotein A-IV amyloidosis was not confined only to the medullary region, but also involved the cortex, and fibrinogen-α amyloid was not confined only to glomeruli but also involved the interstitium and arteries. Accurate diagnosis and typing of amyloidosis is extremely important for treatment and prognosis [10]. It prevents unnecessary chemotherapy and bone marrow transplantation, which can occur if it is wrongly concluded that the amyloid, although not staining for κ or λ light chains, is due to abnormal paraprotein production caused by a subtle plasma cell dyscrasia. In summary, LECT2 amyloidosis is rare. Immunohistochemical study for LECT2 is sensitive and specific, but there is a high false-positive rate if the threshold for immunostaining positivity is not set at an appropriate high level. The data from our study suggest that LECT2 immunohistochemistry may be used along with LMD/MS to avoid inaccurate diagnosis.

References [1] von Hutten H, Mihatsch M, Lobeck H, Rudolph B, Eriksson M, Röcken C. Prevalence and origin of amyloid in kidney biopsies. Am J Surg Pathol 2009;33:1198-205.

P. Paueksakon et al. [2] Markowitz GS. Dysproteinemia and the kidney. Adv Anat Pathol 2004;11:49-63. [3] Sethi S, Thesis JD, Quint P, Maierhofer W, et al. Renal amyloidosis associated with a novel sequence variant of gelsolin. Am J Kidney Dis 2013;1:161-6. [4] Benson MD, James S, Scott K, Liepnieks JJ, Kluve-Beckerman B. Leukocyte chemotactic factor 2: a novel renal amyloid protein. Kidney Int 2008;74:218-22. [5] Larsen CP, Walker PD, Weiss DT, Solomon A. Prevalence and morphology of leukocyte chemotactic factor 2-associated amyloid in renal biopsies. Kidney Int 2010;77:816-9. [6] Murphy CL, Wang S, Kestler D, et al. Leukocyte chemotactic factor 2 (LECT2)–associated renal amyloidosis: a case series. Am J Kidney Dis 2010;56:1100-7. [7] Yamagoe S, Akasaka T, Uchida T, et al. Expression of a neutrophil chemotactic protein LECT2 in human hepatocytes revealed by immunochemical studies using polyclonal and monoclonal antibodies to a recombinant LECT2. Biochem Biophys Res Commun 1997;237:116-20. [8] Nagai H, Hamada T, Uchida T, Yamagoe S, Suzuki K. Systemic expression of a newly recognized protein, LECT2, in the human body. Pathol Int 1998;48:882-6. [9] Vranna JA, Gamez JD, Madden BJ, Thesis JD, Bergen HR, Dogan A. Classification of amyloidosis by laser microdissection and mass spectrometry based proteomic analysis in clinical biopsy specimens. Blood 2009;114:4957-9. [10] Sethi S, Vrana JA, Thesis JD, et al. Laser microdissection and mass spectrometry based proteomics aids the diagnosis and typing of renal amyloidosis. Kidney Int 2012;82:226-34. [11] Leung N, Nasr SH, Sethi S. How I treat amyloidosis: the importance of accurate diagnosis and amyloid typing. Blood 2012;120:3206-13. [12] Picken MM, Herrera GA. The burden of ‘sticky’ amyloid. Typing challenges. Arch Pathol Lab Med 2007;131:850-1. [13] Murphy CL, Wang S, Williams T, Weiss DT, Solomon A. Characterization of systemic amyloid deposits by mass spectrometry. Methods Enzymol 2006;412:48-62. [14] Murphy CL, Eulitz M, Hrncic R, et al. Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens. Am J Clin Pathol 2001;116:135-42. [15] Sethi S, Thesis JD, Leung N, et al. Mass spectrometry-based proteomic diagnosis of renal immunoglobulin heavy chain amyloidodid. Clin J Am Soc Nephrol 2010;5:2180-7. [16] Sethi S, Thesis JD, Shiller SM, et al. Medullary amyloidosis associated with apolipoprotein-IV deposition. Kidney Int 2010;8:201-6.

Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining.

We investigated the role of leukocyte chemotactic factor (LECT2) immunohistochemical staining in the diagnosis of type of renal amyloidosis. Fifty ren...
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