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Localized, paucicellular proteinaceous deposits are occasionally encountered in biopsies of the nervous system. In some instances, the nature of these deposits may be elucidated. Their prototype is found in the amyloidoma, a localized accumulation of AL λ-light-chain-derived amyloid with a predilection for involvement of white matter, choroid plexus, and peripheral nerve.1, 2, 3, 4 Amyloidomas are indolent lesions, and the diagnosis can be confirmed with both histochemical stains for amyloid (Congo red (CR) and sulfated Alcian blue) and immunohistochemistry for κ- and λ-immunoglobulin light chains. Amyloid may also be seen in dural deposits of extranodal marginal zone lymphomas.5

Other light-chain-derived, non-amyloid deposits are known to occur, predominantly in extraneural sites, and usually in association with monoclonal lymphoplasmacytic disorders. The latter include light-chain deposition disease and crystal-storing histiocytosis.6, 7, 8, 9 However, nervous system involvement by these two processes is not well characterized.

Laser microdissection of formalin-fixed paraffin-embedded tissue (FFPE), in combination with liquid chromatography and tandem mass spectrometry (LC-MS/MS), represents a technique with potential as a novel diagnostic method in surgical pathology. We hypothesized that proteinaceous deposits encountered in biopsies of the nervous system, whether demonstrating amyloid properties or not, have immunoglobulin components. In the current study, we explore the utility of laser microdissection in combination with LC-MS/MS for the evaluation of proteinaceous deposits in FFPE in this setting, and provide a rationale for their implementation as useful adjuncts to immunohistochemistry in diagnostic pathology laboratories.

MATERIALS AND METHODS

Patients

The Mayo Clinic pathology records were searched for cases of amyloid, other proteinaceous material deposition (non-amyloid extracellular deposits and intracellular crystals) in brain, spinal cord, or peripheral nerve. A total of 13 (of 15) cases were included in this study. Two cases were excluded because only unstained charged slides were available for analysis. Twelve of the thirteen cases were biopsies, and 1 case was obtained from autopsy (case 9). All slides were reviewed by at least two neuropathologists (CG, JEP, FJR, BWS) and one hematopathologist (AD). Clinical data, demographics, and follow-up information were obtained from retrospective chart review and consultation correspondence. This study was approved by the Institutional Review Board at the Mayo Clinic.

Specimen Preparation and Microdissection

The duration of storage for the FFPE tissues used in this study was variable, with a range of 6 months to 10 years. Thick (10 μm) sections were placed on DIRECTOR™ slides (Expression Pathology, Gaithersburg, MD, USA) for ease of laser microdissection. The tissue was dissected directly from a charged slide in cases where paraffin blocks were not available. At least three different areas were separately microdissected and analyzed in all cases. Sections were air dried and then melted, deparaffinized, and stained in hematoxylin followed by CR. Fluorescence microscope optics was used to identify areas of CR positivity. In the non-amyloid cases the areas of interest were microdissected under bright-field microscopy into 0.5 ml microcentrifuge tube caps containing 10 mM Tris/1 mM EDTA/0.002% Zwittergent 3–16 (Calbiochem, San Diego, CA, USA) using a Leica DM6000B Microdissection System (Wetzler, Germany). Collected tissues were heated at 98°C for 90 min with occasional vortexing. Following 60 min of sonication in a waterbath, samples were digested overnight at 37°C with 1.5 μl of 1 μg/ml trypsin (Promega, Madison, WI, USA).

Protein Identification Via Mass Spectrometry

The trypsin generated digests were reduced with dithiothreitol and separated by nanoflow liquid chromatography–electrospray tandem mass spectrometry using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron, Bremen, Germany) coupled to an Eksigent nanoLC-2D HPLC system (Eksigent, Dublin, CA, USA). A 0.25 μl trap (Optimize Technologies) packed with Michrom Magic C-8 was plumbed into a 10-port valve. A 75 μm × ∼15 cm C-18 column was utilized for the separation utilizing an organic gradient from 6 to 86% in 55 min at 400 nl/min.

The Thermo-Fisher MS/MS raw data files were submitted to an in-house developed workflow tool.10 Three search algorithms (Sequest, Mascot and X!Tandem) were searched and the results assigned peptide and protein probability scores. The results were then displayed in Scaffold (Proteome Software, Portland, OR, USA). All searches were conducted with variable modifications and restricted to full trypsin-generated peptides allowing for two missed cleavages. Peptide mass search tolerances were set to 10 p.p.m. and fragment mass tolerance to ±1.00 Da. The human SwissProt database was utilized (24 July 2007). Protein identifications were confirmed at the 90% confidence level and required two peptides for identification.

Immunohistochemistry

Immunohistochemical stains were performed with the aid of a DAKO Autostainer (Dako North America Inc., Carpinteria, CA, USA) using the Dual Link Envision+ or ADVANCE (Dako) detection systems. Antibodies were directed against the following antigens, with the corresponding clones for the monoclonal antibodies specified: CD3 (Novocastra, Newcastke, UK; clone PS1; dilution 1:50), CD20 (Dako; clone L26; dilution 1:60), CD68 (Dako, clone KP-1; dilution 1:3000), CD138 (Dako; clone M115; dilution 1:50), Kappa Free Light Chains (Dako, polyclonal, dilution 1:6000), Kappa Light Chain (Auto ProEnzyme pretreatment; Dako; polyclonal; dilution 1:2500), Lambda Free Light Chains (Dako; polyclonal; dilution 1:2000), Lambda Light Chain (Auto ProEnzyme pretreatment; Dako; polyclonal; dilution 1:3000), Prealbumin (Transthyretin) (Dako; polyclonal; dilution 1:5000), Serum Amyloid A (SAA; Dako; clone MC-1; dilution 1:1000), and Serum Amyloid P (SAP; Biocare; polyclonal, dilution 1:20).

In Situ Hybridization

In situ hybridization with probes targeting λ- and κ-light chain mRNA was performed on case 7 using Ventana Benchmark XT platform (Ventana, Tucson, AZ, USA) and commercially available probes (Inform® cytoplasmic kappa and lambda probes, Ventana) according to the manufacturer's instructions.

RESULTS

Clinical Findings

The clinical findings are summarized in Table 1. The depositions involved brain (n=12) or peripheral nerve (n=1). The patients included nine women and four men. They were all adults with a median age at diagnosis of 51 years (range, 31–72). Radiologic studies demonstrated multifocal lesions in the majority of the cases, usually involving cerebral white matter (n=6). A single abnormality/mass was present in five cases. In the single case involving peripheral nerve, a localized enhancing enlargement of the sciatic nerve was evident. No imaging data were available for the remaining case.

Table 1 Protein deposits in clinical specimens of the nervous system: clinical features
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Pathology

Histologically, the proteinaceous deposits could be placed within one of the following categories: extracellular proteinaceous deposit not otherwise specified (PDNOS; n=6), amyloidoma (n=5), or intracellular crystals (n=2). The pathologic features are summarized in Table 2.

Table 2 Protein deposits in clinical specimens of the nervous system: pathologic features
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Extracellular proteinaceous deposit not otherwise specified

These consisted of amorphous, flocculent, CR-negative extracellular eosinophilic proteinaceous aggregates with occasional calcification. In two cases well-formed extracellular crystals were also present. Two cases were associated with a contiguous low-grade B-cell lymphoma with lymphoplasmacytic differentiation and demonstrated a foreign body giant cell reaction to the deposits (Figure 1). A marginal zone lymphoma of the spleen was identified at autopsy in one dramatic case characterized by deeply eosinophilic immunoglobulin lakes mostly in white matter but with smaller aggregates in the cortex (Figure 2).

Figure 1
figure 1

Extracellular proteinaceous deposition with crystals (case 6). Aggregates of eosinophilic proteinaceous deposits with crystal formation are surrounded by a dense neoplastic lymphoplasmacytic infiltrate (a). Immunohistochemical stains demonstrate κ-light chain (b) immunostaining but absent λ (c) consistent with κ-light-chain restriction. The latter was also demonstrated by mass spectrometry, which in addition revealed Ig-γ and Ig heavy chain peptides (not shown).

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Figure 2
figure 2

Extracellular proteinaceous deposition in the brain in a case of splenic marginal zone lymphoma. Lakes of eosinophilic deposits lacking crystals are involving predominantly white matter (a), although scant smaller deposits are also present in gray matter (b). Immunohistochemical stains demonstrate κ-light-chain expression (c) but not λ (d), as well as IgM (e).

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Amyloidoma

Amorphous pink aggregates of amyloid were present in a parenchymal and/or perivascular pattern (Figure 3a). CR staining was positive in all cases, showing strong red fluorescence (Figure 3b and c). An associated mild, generally perivascular lymphoplasmacytic infiltrate was also identified.

Figure 3
figure 3

Amyloidoma of sciatic nerve and laser microdissection (case 12). Localized tumefactive amyloid deposit replacing a peripheral nerve fascicle demonstrated on H&E (a) and Congo red (b) stains. Rhodamine optics demonstrates bright red fluorescence (c, Congo red). Several areas are traced in the computer screen, microdissected and submitted for analysis using available software. λ-Light-chain restriction was demonstrated by immunohistochemistry and mass spectrometry (not shown).

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Intracellular crystals

Intracellular crystals were the hallmark in two cases. In case 7, there was a massive intracellular accumulation of needle shaped to rhomboid crystals within CD68+ macrophages throughout the biopsy consistent with intracerebral crystal-storing histiocytosis (Figure 4). Scattered, mostly perivascular plasma cells were present. In case 2, thicker rhomboid crystals were noted within CD138+ plasma cells, which were less frequent than in case 7. ‘Touton-like’ multinucleated giant cells were also present.

Figure 4
figure 4

Crystal-storing histiocytosis (case 7). Intracellular refractile crystals are evident on H&E stain (a). The histiocytic nature of the cells is confirmed by strong CD68 expression (b). In situ hybridization demonstrates κ mRNA (but not λ) focally in perivascular plasma cells (c and d, respectively).

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Mass Spectrometry and Immunohistochemistry

Proteomic analyses provided reliable MS and MS/MS spectra in a representative case (Figure 5), and the Scaffold™ search algorithm provided reproducible protein profiles in all 13 cases tested (Supplementary Data).

Figure 5
figure 5

Tandem mass spectrometry (MS) analysis identifies κ-light chain V-III in case 9. The Orbitrap MS survey scan containing a parent ion mass of 916.943 Da is shown in (a). The LTQ MS/MS scan of the doubly charged ion at 916 Da is shown in (b). A database search against the MS/MS spectra revealed this to be ASQSVSSYLAWYQQK with an XCorr of 5.14 and −1.9 p.p.m. mass error corresponding to Ig κ-light chain (KV312_HUMAN) (c).

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Extracellular proteinaceous deposit not otherwise specified

Interestingly, the non-amyloid extracellular depositions contained κ-light chains, but not λ, in most cases (n=5). One case contained κ- as well as λ-light chains. Additional proteins identified included immunoglobulin (Ig) G (n=5), IgM (n=2), IgA (n=2), IgJ (n=2), apolipoprotein A-I (ApoA-I) (n=3), and ApoE (n=2). Ig heavy chain, ApoA-II, and C9 were identified in single cases each. Immunohistochemistry with κ- and λ-light chain confirmed κ-light chain restriction in four cases, either in the deposits (n=3) and/or the inflammatory infiltrate (n=2). A polytypic pattern of light-chain expression by immunohistochemistry was present in the single case demonstrating both κ- and λ-light-chain peptides by LC-MS/MS. In addition, one (of two) cases with IgM showed convincing immunostaining (Figure 2).

Amyloidomas

λ-Light chains, SAP, ApoE, and ApoA-IV were identified in all cases (n=5). In addition, ApoA-I was identified in three cases, and IgG and IgA in single cases each. Immunohistochemistry demonstrated λ (but not κ) light chains in the aggregates in all cases, and SAP in two (of three) cases tested.

Intracellular crystals

The example consistent with crystal-storing histiocytosis (case 7) demonstrated κ-light chain as well as a minor GFAP component on LC-MS/MS. The GFAP was likely secondary to the presence of underlying brain tissue/reactive astrocytes in the microdissected area. Immunostains for κ- and λ-light chains were non-contributory secondary to ample background staining. However, in situ hybridization labeled monotypic κ-plasma cells (Figure 4). Conversely, in case 2, IgG was identified by LC-MS/MS. The crystals demonstrated reactivity with IgG and λ-light chains, but not κ, antibodies by immunohistochemistry.

DISCUSSION

Protein characterization in tissue samples is an active field that has generated much excitement in recent years. Protein analysis focused on identification of single proteins or peptides has been most extensively explored in serum using gel electrophoresis/western blotting or immunoassays, such as ELISA.11, 12 However, some of these commonly used assays are limited in their sensitivity, require relatively large amounts of sample, and focus on only a small number of proteins. These traditional methods have also been successfully applied to cell lines and fresh or frozen tissue for protein analyses, but are not applicable to FFPE tissues.

Immunohistochemistry is a technique that has been studied and extensively applied for protein identification in diagnostic pathology and research. Although presently it is the most widely applied technique to study protein expression in FFPE archival material, immunostaining of extracellular protein deposits is problematic as antigenic epitopes are often lost in the tertiary structure of the deposit, and contamination from serum proteins often gives rise to marked background that yields indeterminate results. Formal doubts have been raised about the reliability of immunohistochemistry in determining the amyloidosis type.13 Furthermore, these studies are limited by the small number of markers that can be simultaneously studied in a single tissue section, as well as the variety and number of commercial antibodies available. Of the methods discussed, only immunohistochemistry has been extensively used in the study of FFPE tissue. Although FFPE is the primary archival form of pathologic specimens, and permits retrospective study of numerous samples with clinical follow-up, the extension of high throughput analyses to this resource should yield a wealth of biologic information.

With the advent of proteomics, newer technologies have emerged that bypass all these limitations. In addition to its promise as a powerful diagnostic technique that is the subject of the current study, MS may be considered at the present time the technique with the greatest potential for unbiased biomarker discovery, as well as significantly aiding in our understanding of disease pathogenesis.14 High throughput platforms that utilize MS have been successfully applied to serum peptide profiling. Examples include its application to glioblastoma,15 as well as ovarian16 and prostate carcinoma.17 Other applications include mapping of plasma membrane proteins in brain18 and the detection of DNA-mismatch repair-deficient cells.19 Combining MS with immunoaffinity enrichment has also been successful in identifying chromosomal translocation partners in anaplastic lymphoma.20

Traditionally, the application of proteomics to FFPE has been limited by several factors, including the crosslinks induced by formaldehyde and alterations secondary to the embedding process that affects protein solubility. However, some authors have been successful in extending proteomic analyses to these tissues with appropriate methods that involve proteolytic cleavage,21, 22, 23 including in situ enzymatic digestion and even matrix-assisted laser desorption/ionization MS,24 which allows for delineation of the spatial distribution of proteins. A proteomic-based approach to surgically obtained samples complements the study of gene expression profiling, given the greater susceptibility of RNA for degradation, as well as the possibility to study post-translational modifications25 difficult to investigate with traditional genomics.

We hypothesized that the application of MS to protein analysis may not be restricted to proteomic research, but that it may also be of use in diagnostic surgical pathology. We have found that sample digestion with separation of peptide mixtures by chromatography followed by analysis in a modern hybrid mass spectrometer produces accurate information about the composition of complex samples and is feasible to perform in archival material. Laser microdissection, a technique with numerous applications in molecular analysis,26, 27 enhances diagnostic precision in this setting. A probability-based algorithm and comparison with available databases greatly simplifies and increases the efficiency of protein characterization.28

In our experience, proteinaceous deposits encountered in biopsies of the nervous system often represent diagnostic challenges even after a thorough clinicopathologic work-up. Among these, amyloidomas have been best characterized, although it is known that other forms of light-chain-derived deposits that lack the ultrastructural and histochemical properties of amyloid may deposit systemically as in light-chain deposition disease,6 or in the form of tumefactive masses.29, 30 Systemic light-chain deposition disease31 as well as reported tumefactive extraneural aggregates thereof29, 30 and crystal-storing histiocytosis7, 8, 9 shows a tendency to κ-light-chain restriction. It is of interest that two previously reported cases of light-chain deposition disease involving the brain were λ-light chain derived,32, 33 unlike our cases in which non-amyloid deposits were mostly κ. Of note neither of those two previously reported cases was evaluated by MS. Our present study extends the use of MS to the characterization of these deposits in FFPE. There was an excellent concordance with immunohistochemical results, although MS confirmed light-chain restriction and the nature of intracellular deposits in at least one case with non-contributory immunostaining.

A point of practical importance involves the concurrence of a lymphoplasmacytic proliferation with a specific type of protein deposit. In cases with amyloid deposition, as those described in the literature, an associated lymphoproliferative disorder is not present, with the exception of extranodal marginal zone lymphoma of the dura. Conversely, in cases of PDNOS, a low-grade B-cell lymphoma with plasmacytic differentiation may be an identifiable etiology. As demonstrated in one of our patients with a splenic marginal zone lymphoma, the neoplastic process may not be evident in the brain lesion. Such cases require a careful systemic work-up and close follow-up to discover the neoplasm. The demonstration of non-amyloid proteinaceous deposits containing κ-light chain may, therefore, be of clinical and prognostic importance.

A further illustrative aspect of our study is that MS found these various protein deposits to be complex, including additional immunoglobulin and apolipoprotein-derived peptides. It is known that a variety of apolipoproteins including ApoA-I, ApoA-II, and ApoA-IV are amyloidogenic, a phenomenon that is enhanced by specific alleles. This has been studied in transgenic mice models.34 Several of these proteins were identified in our analyses. These proteins are not routinely evaluated on amyloid immunohistochemical panels. Although they seem to be involved in amyloid formation, it is unclear what their role is in non-amyloid deposits. Our limited study does not provide any mechanistic insights into pathogenesis, but future studies with a larger number of cases, and more detailed exploration of the significance of the various peptides identified, should further our current limited understanding of these disorders.

In summary, proteomics is beginning to have an important role, not only in research into human disease, but also in molecular diagnostics as well. The use of novel exquisitely sensitive and specific practical techniques, including MS, in the analysis of protein accumulations enhances the diagnostic precision afforded by routine histology and immunohistochemistry. We employed this technique in a focused study of immunoglobulin-derived deposits, but we believe in its potential to find widespread applications in diagnostic surgical pathology in addition to its better-publicized role in biomarker discovery.