Pathology
Extracellular
Intracellular
Intra-vascular
Organized
Non-organized
Renal
Extrarenal
Light chain cast nephropathy
+a
Amorphous
+
−
AL/AH amyloid
+
Fibrillar Congo red (+)
+
S/L
MIDD
+
Powdery
+
S
LCPT
+
Crystalline
Noncrystalline
+
−
CSH/ISH
+
Crystalline
Noncrystalline
+
S/L
Cryoglobulinemia
+/−
+
Focallyb
+
Skin/S?
Waldenström macroglobulinemia
+
+
+
Skin/S?
Crystalglobulinemia
+
+
Skin/S?
GN with monoclonal deposits
+
Electron dense
+
?
Immunotactoid GN
+
Microtubules
+
?S
Paraprotein-associated tubulointerstitial GN/c
+/−?
+/−?
+/−?
+/−?
+/−?
+
?
While pathology associated with light chain cast nephropathy affecting distal tubules is frequent and a well-known complication of paraproteinemia, proximal tubule injury is less frequently reported, less well known, and, most likely, underreported. Currently, only approximately 110 cases have been reported that deal with this subject. The most frequently used terms include adult Fanconi syndrome (FS), light chain Fanconi syndrome (LCFS), crystalopathies, and/or light chain proximal tubulopathy (LCPT) [1, 2, 6–11] (see Figs. 12.1, 12.2, 12.3); proximal tubular pathology may be associated with renal interstitial and/or systemic crystal-storing histiocytosis ([12–14], see also below).
Fig. 12.1
Paraffin section showing rare crystals in the proximal tubular epithelial cells. There is also a striking granularity of the cytoplasm (hematoxylin–eosin, original magnification ×200)
Fig. 12.2
Immunofluorescence stain for kappa light chain limited to the proximal tubular epithelium. No deposits are detectable by immunofluorescence in the adjacent glomerulus. Stains for lambda light chains, immunoglobulin (Ig) G, IgA, IgM, and complements were negative (not shown) (original magnification ×400)
Fig. 12.3
(a) Electron micrograph showing abundant rhomboid crystals within the proximal tubular epithelium (uranyl acetate stain, original magnification ×3000). (b) Electron micrograph showing the lattice-like structure of the rhomboid crystals (uranyl acetate stain, original magnification ×50,000)
Since 1954, it has been known that adult patients may develop acquired FS in association with PCD as a consequence of acquired proximal tubular injury [6]. In 1975, Maldonado et al. reviewed the first large series of 17 (new and previously published) cases of FS with Bence Jones proteinuria and myeloma or amyloidosis [7]. In most cases, the diagnosis of FS preceded the development of myeloma or amyloidosis, but not the reverse, while, in some patients, the diagnosis of FS and multiple myeloma or amyloidosis was established simultaneously. There was a relatively indolent renal dysfunction, Bence Jones proteinuria of κ-type, and, in some cases, osteomalacia resulting from chronic hypophosphatemia. Kidney biopsy showed prominent crystals in proximal tubular epithelium and similar crystals were also seen in the bone marrow in many patients. No myeloma casts were seen in the distal tubules. The authors could not demonstrate serum protein monoclonal abnormalities. Based on this initial series, it was proposed that patients with Fanconi syndrome and Bence Jones proteinuria have a distinct type of plasma cell disorder or a variant of the monoclonal gammopathies, termed LCFS, characterized by slow progression of the tumor and an early phase dominated by the metabolic complications of renal proximal tubular dysfunction.
In 1993, Aucouturier et al. sequenced the κ-cDNA from a patient with LCFS and demonstrated that it belonged to the VK1 subgroup and was characterized by resistance to complete proteolysis and the ability to form crystals [8]. In contrast, the light chains from 12 patients with myeloma cast nephropathy were susceptible to proteolytic digestion. The propensity of light chains in LCFS to form crystals that precipitate within the cytoplasm of proximal tubules appears to be determined by changes in their amino acid sequences. The circulating light chains are filtered through the glomerular capillary wall and bind to the luminal surface of the proximal tubular epithelial cells; subsequently, the light chains are incorporated into endosomes, which fuse with lysosomes, where they are then degraded into amino acids and returned to the circulation. While the “non-pathogenic” light chains are completely degraded, in FS there is incomplete proteolysis, which leads to the accumulation of protease-resistant fragments that constitute a nidus for crystal formation. Thus, the development of LCFS appears to be associated with the common origin of the fragments—from the light chain VK1 subgroup—and their primary sequence peculiarities, which lead to partial resistance to proteolysis with the formation of a truncated NH2-terminal fragment with a propensity to crystallize. In 2006, Sirac et al. published a transgenic murine model of LCFS, which supported the above-proposed pathogenesis of this condition [9]. Subsequent research by El Hamel et al. [14] and Toly-Ndour et al. [15] extended the molecular studies, further supporting the evidence that amino acid substitutions, including distinct hydrophobic residues, favor the formation of crystals. Specific impairment of proximal tubular cell proliferation by a monoclonal κ-light chain responsible for FS was demonstrated by El-Hammel et al. in 2012 [16]. However, the mechanism(s) leading to FS (glycosuria, phosphaturia, aminoaciduria) is/are poorly understood and, indeed, some patients may have only partial FS or no FS (reviewed by Sirac et al. [17–19]).
With subsequent publications, the clinical picture associated with injury to the proximal tubular epithelium was expanded and the associated pathologic spectrum was increased to include non-organized intracellular paraprotein deposits as well [11, 20]. In 2007, the authors (MMP et al.) published a report of five cases of light chain proximal tubulopathy and broadened its definition to include three cases in which electron microscopy revealed only prominent phagolysosomes within tubular epithelia; by immuno-electron microscopy, these phagolysosomes contained a single light chain, kappa or lambda [11] Thus, the authors concluded that some patients with PCD may have more subtle evidence of proximal tubular injury not associated with crystals but only with lysosomal light chain restriction. This restriction can be detected by immunofluorescence as well as by immuno-EM. The light microscopic morphology may be quite subtle and only suggestive of acute tubular injury. To encompass this expanded definition, the authors proposed the term “light chain proximal tubulopathy” (LCPT) [11] (Fig. 12.4).
Fig. 12.4
Immunogold stain for lambda light chain, which is positive within phagolysosomes in the proximal tubular epithelium. The stain for kappa light chain was negative (not shown) (original magnification ×12,000). (Figures 12.1–12.4. reprinted with permission from Kapur U, Barton K, Fresco R, Leehey DJ, Picken MM. Expanding the pathologic spectrum of immunoglobulin light chain proximal tubulopathy. Arch Pathol Lab Med. 2007 Sep;131(9):1368–72)
Subsequent studies suggested that LCPT not associated with crystals was more frequent than LCPT with crystals. In 2011, Larsen et al. reported ten cases of LCPT without crystals, which constituted 3.1 % of their light chain-related diseases, while, in their material, only three cases (0.9 %) had crystals [20]. Similar results were reported by others [21–23]. In Herrera’s series of 57 cases with proximal tubulopathies, only four biopsies showed crystalline inclusions [23]. At the molecular level, the absence of crystals is often associated with normal sensitivity to proteolysis, suggesting that reabsorption of monoclonal light chains might also interfere with proximal tubule function through a direct toxic effect [17]. To this end, it is postulated that excessive endocytosis of the light chains promotes apoptosis and tubular damage.
In previous years, most cases of LCPT have been reported with kappa light chain, while only rare cases of LCPT were associated with λ-light chains (with or without crystals) [1, 2, 6–8, 10–31]. However, more recent reports indicate that lambda light chains may be more often associated with LCPT than kappa light chains [11, 20, 21]. While kappa light chains are preferentially associated with crystals, lambda light chains are typically associated with noncrystalline LCPT. Thus, in Larsen’s series, nine out of ten biopsies with LCPT without crystals showed λ-light chain restriction [20].
The prognosis is variable and largely depends on the tumor burden [11, 32]. However, the diagnosis of LCPT is of critical importance since this condition is often associated with previously unrecognized myeloma/PCD/B-cell lymphoplasmacytic disorder [11, 20, 23, 33]. Moreover, tubular injury progresses over time leading to renal failure.
Pathology
Light microscopic findings are largely nonspecific and suggestive of acute proximal tubular injury and/or chronic tubulointerstitial nephropathy [11–31]. Rare tubular epithelial cells may contain pale needle-shaped crystals that are mainly visible at high magnification [11, 23, 33] (Fig. 12.1). These crystals are PAS negative and may stain red or green with trichrome stain. At times, in cases where no crystals are discernible by light microscopy, there may be a diffuse proximal clear cell transformation resulting in a pseudo-osmotic nephrosis pattern [33, 34]. Some cells exhibit only prominent cytoplasmic granularity, or a glassy appearance, vacuolization, cellular enlargement (“hypertrophy”), loss of brush border, and sloughing, resulting in an acute tubular necrosis-like picture [11, 14, 23, 33, 34].
Based on morphology, Herrera recently subdivided LCPT into four categories including (1) proximal tubulopathy without cytoplasmic inclusions, (2) tubulopathy associated with interstitial inflammatory reaction, (3) proximal tubulopathy with cytoplasmic inclusions, and (4) proximal tubulopathy with lysosomal indigestion/constipation; each category was represented by 22, 28, 4, and 3 cases, respectively, in his series of 57 patients [23].
Crystal deposition in the mesangial cells [35] or in podocytes with glomerular dysfunction [36] and isotypic light chain cast nephropathy has also been reported [13, 37]. In one recently reported case, histiocytes with lysosomal accumulation of λ-light chain and absence of κ-light chain were seen within the glomerular capillaries [38]; segmental endocapillary proliferative glomerulonephritis was also visible.
In some patients, infiltration by mononuclear cells with interstitial fibrosis may dominate the light microscopic pathology. The presence of intracytoplasmic crystals in the proximal tubular epithelium may be associated with the presence of crystals in the interstitial macrophages in the kidney and/or of the bone marrow and/or other organs (termed crystal-storing histiocytosis; see below and [13, 14, 39]).
By immunofluorescence, in most (but not all) cases, there is light chain restriction, either for kappa or lambda light chain, which is limited to proximal tubular epithelium [11, 20, 23] (Fig. 12.2). Interestingly, in rare cases, crystals were negative by standard immunofluorescence performed on frozen tissue, while immunofluorescence on formalin-fixed, paraffin-embedded, pronase-digested tissue showed positivity for a single light chain [33]. It is proposed that, with pronase digestion, a denaturing effect on cell membranes is achieved, which may facilitate access of the antibody to the lysosome-bound crystals and, possibly thereby, unmask sequestered antigenic sites.
By electron microscopy, abundant crystals of varying sizes and shapes are seen [11, 20, 23, 33] (Fig. 12.3a and b). These crystals may be rectangular, rhomboid, round, or needle-shaped and are surrounded by a single membrane, most likely of lysosomal origin. Similar crystals in the urinary sediment have also been reported [14]. Interestingly, there is a certain range of ultrastructural morphology of crystals, including hexagonal or diamond-shaped crystals with a lattice-like structure in some, and needle-shaped, rectangular, and rhomboid patterns in others. El-Hammel et al. suggested that these differences may result from distinct structures of monoclonal light chains [14]. Moreover, in one of their patients with Fanconi syndrome, crystals showed a fibrillar amyloid-like organization, which, by immuno-electron microscopy, strongly reacted with anti-kappa light chain antibody [14]. A microtubular or fibrillary amyloid-like structure of inclusions was also reported earlier by Taneda et al. [40] and Herlitz et al. [33], Yao et al. [41], and Corbett et al. [42]. In Herrera’s series, among four cases of LCPT with inclusions, in one case electron microscopy demonstrated a fibrillary rather than a crystalline appearance [23]. However, the authors provided no information as to whether these inclusions were Congo red positive and birefringent when viewed under polarized light, which would fully qualify them as intracellular amyloid. However, in 2012, two reports of an apparent amyloid proximal tubulopathy were published [43, 44]. In both cases, rare proximal tubular cells showed a distinct Congo red-positive inclusion, which were birefringent under polarized light (Fig. 12.5). In case reported by Hemminger et al., intracellular amyloid formation was associated with concomitant phenotypic changes, suggestive of histiocytic differentiation of tubular epithelial cells [44].
Fig. 12.5
Amyloid LCPT: (a) Several individual cells show apple-green birefringence. Congo red-stained slide viewed under polarized light, (b and c) Focal intracytoplasmic, round, and homogeneous inclusions are pale blue in trichrome-stained section (b) and amphophilic in H&E-stained sections. Notice interstitial nephritis-like component with lymphocytes and occasional eosinophils (slides courtesy of C. Larsen, photographed and published with permission)
In biopsies without crystals, there are abundant and often enlarged and bizarre phagolysosomes, which show light chain restriction by immuno-electron microscopy and/or immunofluorescence [11, 20, 23] (Fig. 12.4). In some patients, such lysosomal inclusions in the proximal tubular epithelium have been associated with crystal-bearing histiocytes infiltrating the renal interstitium [14].
Clinical picture. While an underlying PCD is largely unsuspected prior to kidney biopsy, following the diagnosis of LCPT, virtually all patients are found to have some form of PCD, including MGUS, multiple myeloma (“smoldering multiple myeloma”), or, less commonly, isotypic amyloidosis; some patients may have lymphoplasmacytic lymphoma [7, 10–36]. The clinical picture may be subtle and diverse, depending on tumor burden [7, 11, 18, 20–23, 32]. Nevertheless, even in cases associated with low tumor burden and a seemingly indolent clinical course, kidney failure develops [11]. Interestingly, kidney failure can be reversed upon elimination of the abnormal protein ([33, 39], Picken—unpublished observation). Reversibility of LCPT has also been supported by animal studies [9]. If untreated, LCPT can recur in kidney transplant patients [11]. Given that LCPT leads to renal failure, this suggests that it should be treated to suppress the production of nephrotoxic monoclonal light chains [39, 45]. Some preliminary studies suggest that novel immunomodulating agents, as well as direct removal of the light chains by plasma exchange or intensive dialysis using high cutoff membranes, may improve renal prognosis in LCPT ([39, 45, 46], Picken—unpublished observation).
Thus, adult patients with Fanconi syndrome should be carefully investigated for plasmacytic dyscrasia/B-cell lymphoproliferative disorder; similarly, a pathologic diagnosis of LCPT should prompt a thorough hematologic workup. LCPT may be underdiagnosed because of limited awareness of the entity. In Herrera’s series, which is largest to date, the 57 cases of proximal tubulopathy represented approximately 1.5 % of all patients. However, among lesions related to PCD, proximal tubulopathies were diagnosed in approximately 46 % of all cases [23].
Extrarenal Pathology Associated with Intracellular Paraprotein Storage: Crystal-Storing Histiocytosis (CSH) and Beyond
CSH is a reactive histiocytic hyperplasia in which the histiocytes contain intralysosomal prominent, crystalline, cytoplasmic, immunoglobulin inclusions [12, 14, 47–66]. CSH occurs in plasma cell/B-cell lymphoproliferative disorders. The neoplastic plasma/B-cells may also contain prominent immunoglobulin inclusions and show predominantly kappa light chain restriction. Most reported cases of CSH are associated with multiple myeloma and other PCD or low-grade B-cell lymphomas such as lymphoplasmacytic lymphoma, and marginal zone B-cell lymphoma (typically extranodal, associated with mucosa-associated lymphoid tissue, MALT lymphoma), some of which arise in association with reactive conditions such as Sjögren’s syndrome [55]. Rarely, CSH was reported in apparently inflammatory-reactive processes. However, in these latter cases, the possibility of an undiagnosed low-grade lymphoproliferative process could not be excluded [3, 56, 57]. The crystal deposition can be localized at the site of lymphoplasmacellular proliferation or may be systemic. In systemic CSH, the reticuloendothelial system is involved, with extramedullary sites.
Crystals can be seen either in histiocytes in soft tissues or parenchymal cells. The presence of intracytoplasmic crystals in the macrophages of the bone marrow may be coincident with the presence of similar crystals in renal interstitial macrophages and/or in the proximal tubular epithelium. The crystals usually accumulate within lymphoid cells and hematopoietic tissues (bone marrow, lymph nodes, spleen, thymus) or within epithelial cells and connective tissue stroma of the kidney, lungs, pleural effusion, thyroid, parotid gland, eye structures (cornea, orbital fat, lacrimal gland, eyelid, conjunctiva), skin, subcutaneous fat, heart, testes, tongue, liver, stomach, adrenals, or skull and even brain parenchyma [12, 14, 47–62, 67–80]. Initial presentation largely depends on the localization: some patients present with a soft-tissue mass with abundant histiocytes and fibroblasts containing crystals.
The mechanism of crystal formation and their storage in the histiocytes appears to be similar to that occurring in proximal tubular epithelium [12, 14]. Thus, similar to LCPT, in most reported cases of CSH, kappa light chain was involved, though lambda light chain-associated cases have also occasionally been described; there is no relationship between the types of heavy chains [12, 14, 47, 75, 81]. A single case of plasmacytoma with crystal-storing histiocytosis exhibiting FGFR3 and IgH translocation has recently been published [76].
Thus far only single cases of CSH were examined by laser microdissection (LMD)-tandem mass spectrometry (MS/MS) including three cases of CNS (2 cases [77], 1 case [75]) and two cases of gastric and a single case of nodal CSH [78]. In one case of CNS CSH, associated with monotypic κ-plasma cells by in situ hybridization, LC-MS/MS demonstrated k-light chain. In a second case, IgG was identified by LC-MS/MS [77], while IHC showed reactivity with IgG and λ- light chain. In a third case, LM-MS/MS confirmed the presence of κ-light chain and an IgA constant region [75]. Interestingly, there was an especially high signal for the somatically mutated variable domain fragment of the immunoglobulin κ-light chain [75]. In two cases of gastric CSH, LC-MS/MS demonstrated fragments of the variable domain (V-2) of IgG in one case and fragments of the variable domains (V-1, V-3) of kappa light chain in the second case. In one case of nodal CSH, fragments of variable (V-3) and constant regions of kappa were detected. Variable regions V-1 and V-3 of kappa and the variable region (V-2) of the immunoglobulin heavy chain appeared to have selective representation. These proteomic findings indicate that the crystals are formed by a cleavage product of the immunoglobulin molecule, representing a unique variable region rather than the whole immunoglobulin.
Thus, similar to LCPT, sequence abnormalities at specific sites in the light chain, especially in the kappa light chain, may be responsible for crystal formation as a consequence of defects leading to the loss of a proteolytic site(s) and the generation of fragments with a high intrinsic stability, which then form a nidus for crystallization. Thus, crystallization occurs after endocytosis and proteolysis within the endolysosomal compartment of histiocytes (or epithelial cells in LCPT).
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