Lambda light chain crystalline proximal tubulopathy with probable light chain cast nephropathy and clonal plasma cell infiltrate – uncommon manifestations of a rare form of multiple myeloma

Light chain proximal tubulopathy (LCPT) is an uncommon renal disease characterized by the accumulation of monoclonal light chains within proximal tubular epithelial cells, with or without crystal formation. We report a rare case of lambda LCPT with crystals. Renal biopsy showed substantial acute tubular injury with unusual cytoplasmic changes affecting proximal tubules. In addition, abnormal tubular casts suggested concomitant light chain cast nephropathy. A clonal plasma cell infiltrate was present in the tubulointerstitial compartment. Immunofluorescence demonstrated strong staining for lambda light chain in tubular epithelial cells. Despite the absence of discernible crystals on light microscopy (LM), they were readily identified when ultrastructural evaluation was undertaken. Crystalline inclusions demonstrated positive immunogold labelling for lambda.


Introduction
Renal dysfunction is increasingly recognized in patients with monoclonal gammopathy, resulting from clonal proliferation of B-lymphocytes or plasma cells and subsequent overproduction of monoclonal immunoglobulins. Diseases such as AL amyloidosis, light chain cast nephropathy and monoclonal immunoglobulin deposition disease are some of the more common disorders involving accumulation of abnormal proteins or immunoglobulin deposits in their various forms within the kidney. Light chain proximal tubulopathy (LCPT) is characterized by presence of monoclonal light chains within proximal tubular epithelial cells, with or without crystal formation. It is important that LCPT not be overlooked as an underlying plasma cell dyscrasia may not be suspected clinically at the time of biopsy in many patients (1). In fact, only 0.5% of patients known to have multiple myeloma also had concomitant LCPT in one study (2). The concept of monoclonal gammopathy of renal significance (MGRS) was introduced in 2012 (3) and renal manifestations of a monoclonal gammopathy can be the first signs of an underlying neoplastic hematological condition. Based on a few recent large LCPT case series (1,(4)(5)(6), there were more cases with Journal of Nephropathology, Vol 10, No 1, January 2021 www.nephropathol.com 2 crystals (59.7%) compared to those without. Kappa light chain was detected in the great majority of cases having crystalline inclusions. We report a case of lambda LCPT with crystals, highlighting some unusual light microscopic and ultrastructural features.

Case Presentation
A 60-year-old gentleman of Indian descent presented to Changi General hospital upon referral by his general practitioner for anemia. He had consulted his doctor for dyspnea which had persisted for a few months. A complete blood count found a haemoglobin level of 6.8 g/dL. Prior to this presentation, he had no significant medical history except reflux symptoms, for which an esophagogastroduodenoscopy found nothing untoward. He was treated with a brief course of gastric motility agents and a proton-pump inhibitor. He was normotensive, non-diabetic and had no known renal conditions or any significant family history. Investigations disclosed significant renal impairment with a serum urea of 28.0 mmol/L and creatinine of 741 umol/L (estimated glomerular filtration rate 6 mL/min/1.73 m 2 ). There was no electrolyte derangement and serum total calcium was within the normal range at 2.49 mmol/L. Serum bicarbonate was 12 mmol/L and a gap acidosis was present. This was attributed to uremia as serum lactate and ketones were normal, and no exogenous substances were suspected. Liver function test was normal. Anemia was attributed to chronic kidney disease. Urine microscopy was unremarkable. Urine dipstick revealed 1+ protein and a 24-hour urine collection quantified proteinuria to be 2.78g/day. Ultrasound scan showed normal sized but echogenic kidneys. Further work-up showed positive anti-nuclear antibody at 1:160 dilution (homogenous pattern), although the extractable nuclear antigen panel was negative. Anti-double stranded DNA and antiphospholipase A2 receptor antibodies were also negative. A kidney biopsy was performed for a definitive diagnosis. At the same time, the myeloma screen revealed a normal serum kappa free light chain (FLC) concentration of 17. 11, whilst cytogenetic analysis revealed complex structural abnormalities involving multiple chromosomes. Further, allele specific PCR for Myd88 L265P mutation was negative. PET-CT disclosed multiple areas of increased FDG uptake in the axial and proximal appendicular skeleton, some associated with bony lytic lesions.
The renal biopsy procured two cores of renal cortical and one core of corticomedullary tissue with 44 glomeruli. Three glomeruli (7%) were globally sclerosed. There was focal periglomerular fibrosis. Most of the remainder were fairly unremarkable apart from showing slight capillary wall wrinkling. There was no significant mesangial expansion or hypercellularity. No nodular transformation of mesangial regions was seen. Capillary loops were mostly single contoured, patent and not significantly thickened, devoid of endocapillary hypercellularity and fibrin thrombi. A cellular crescent was present. No necrotizing lesions were found. Occasional glomeruli were involved by NOS-type (not otherwise specified) segmental sclerosis. Mild podocyte hypertrophy was encountered. There were no crystalline inclusions, fuchsinophilic immune deposits or argyrophilic basement membrane spikes.
Acute tubular injury was substantial, with cytoplasmic vacuolation, apical blebbing, epithelial flattening and desquamation. Many proximal tubular epithelial cells were also swollen and packed with pale, vacuolated cytoplasmic inclusions that had a peculiar hue on trichrome stains ( Figures 1A, 1B and 1C). Sloughed, swollen epithelial cells filled the lumen of a number of tubules. No convincing cytoplasmic or luminal crystals were present, despite a thorough search. Several tubules also had PAS-negative casts, at times "fractured" and having sharp edges. These casts appeared polychromatic on trichrome stains ( Figures  2A and 2B). Moderate tubular atrophy with mild but not insignificant interstitial fibrosis was observed. There was moderately extensive lymphoplasmacytic inflammation. Immunostains highlighted a preponderance of CD3+Tlymphocytes and a smaller population of CD20+ B-cells. By in-situ hybridisation, some plasma cells exhibited lambda light chain restriction (Figures 2c and d). No atypical lymphoid cells were discerned. Mild arteriolar hyalinosis and arterial sclerosis were noted. No vasculitis or thrombotic microangiopathy was evident. No congophilic material (amyloid) was detected in any of the renal compartments. Tubular basement membranes were not substantially thickened.
On immunofluorescence microscopy, glomeruli were negative for all immunoreactants, namely IgG, IgA, IgM, C3, C1q, C4, kappa light chain, lambda light chain, fibrinogen and albumin. Tubular basement membranes were also unremarkable. However, proximal tubular epithelial cells stained very intensely for lambda light chain ( Figure 1D Electron microscopic examination showed segmental thickening and wrinkling of the glomerular basement membrane (GBM). Podocyte foot process effacement was significant, affecting at least 60% of peripheral capillary wall surface area, associated with microvillous transformation of podocyte cytoplasm. There were no conventional amorphous or organized non-crystalline deposits in any location including extraglomerular sites. In particular, fine powdery subendothelial and tubular basement membrane deposits were absent, ruling out monoclonal immunoglobulin deposition disease. The cytoplasm of many proximal tubular epithelial cells contained crystalline inclusions, generally rhomboid in shape ( Figure 3A). There were large and abnormally shaped lysosomes in a minority of tubular cells, a number of which had internal crystalline inclusions ( Figures 3B  and 3C). Spillage or release of crystals into the lumina of several tubules was attributable to epithelial cell rupture ( Figure 3D). In the interstitial compartment, scattered histiocytic cells also revealed intracytoplasmic crystals. On higher resolution, some of the crystals displayed internal geometric lucencies or parallel linear arrays ( Figures 3E  and 3F). Immuno-electron microscopy (immunogold    Figure 3F). There was less obvious labelling for this light chain in a number of lysosomes. The Bowman's space of one glomerulus had a small number of crystals.
The patient was commenced on hemodialysis. Chemotherapy comprising daratumumab, bortezomib, cyclophosphamide and dexamethasone was initiated with a view to subsequent autologous stem cell transplantation. He tolerated chemotherapy well and showed symptomatic improvement as well as significant recovery in hemoglobin level and renal function. First disease reassessment showed that the M band had completely disappeared but there was a remarkable increase in serum lambda FLC concentration to >1000 mg/L. Hence, cyclophosphamide was replaced by lenalidomide in his chemotherapy regime.

Discussion
LCPT is a MGRS-defining entity but most patients will not meet the criteria for MM at the time of diagnosis. According to the consensus statement published by the International Kidney and Monoclonal Gammopathy Research Group (IKMG), only 12% to 33% of LCPT cases had underlying MM (7). LCPT is caused by buildup of abnormal light chains in proximal tubular cells. Under normal physiological conditions, FLCs are filtered through the GBM and reabsorbed by proximal tubular epithelial cells through specialized receptors, endocytosed and transported to lysosomes for degradation. However, excess immunoglobulin light chains produced by a clonal population of plasma cells often have mutations in the variable (V L ) region which confer resistance to degradation. As a result, excessive light chains accumulate within the cytoplasm and lysosomes, causing cell injury (8). Lysosomes are thought to play an important role in the processing of light chains. In fact, there have been experiments demonstrating the central role of lysosomes in AL amyloidogenesis through the acquisition of a macrophage phenotype in transformed mesangial cells (9). Supersaturation and unique physicochemical properties predispose to crystallization. In the absence of crystalline inclusions, some cases with excess FLC in tubular cells represent physiologic trafficking rather than target organ damage. Crystalline LCPT involves kappa light chain most of the time, in particular the V k 1 subgroup, with only rare examples showing lambda-restricted intracytoplasmic crystals. Some authors have postulated that V L region mutations introduce additional hydrophobic or apolar amino acid domains to the structure of FLCs, enhancing crystal formation (10,11). The rarity of lambda compared to kappa-restricted disease remains enigmatic. To our knowledge, 9 cases with lambda restriction have been reported in the literature (1,4,11-16). Five had underlying MM, one had smoldering myeloma, one was diagnosed as monoclonal gammopathy of undetermined significance, one case was lymphocytic lymphoma and the underlying hematological condition was unclear in the last case. Many of the crystals seen in our patient's kidney resided in the cytoplasm of proximal tubular epithelial cells, with fewer present in lysosomes. They were mostly rhomboid in shape and had internal geometric lucencies or linear arrays. Additionally, a small number of abnormal or mottled lysosomes were observed next to the crystals. Apart from proximal tubules and histiocytes, light chain crystals have been detected in podocytes, bone marrow plasma cells and even hepatocytes (17).
Changes noted on light microscopy (LM) mirrored those described in the literature. Features of acute tubular injury were rather non-specific but swollen tubular cells with unusual vacuolation and discoloration pointed to the presence of abnormal substances (crystals or lysosomes) within those cells. This case reemphasizes the observation that crystals may not be apparent on LM even if they are conspicuous under electron microscopy. Abnormal lysosomes can give rise to similar cellular changes indistinguishable from crystals on LM. As alluded to previously, abnormal casts resembling light chain or Bence Jones casts were noted only in the LM sample and involved very few tubules. Hence, light chain restriction could not be confirmed in these casts by immunofluorescence. Immunohistochemistry was not pursued as it is not uncommon to have non-specific higher intensity staining for kappa in renal tissue, likely resulting from reduced clearance in the setting of impaired renal function. We believe that as tubular cells became severely damaged, cytoplasmic contents including crystals were released into tubular lumina. Spontaneous crystallization of Bence Jones casts can occur but is rare, as binding to uromodulin and lack of lysosomal processing in the extracellular luminal environment may impede crystal formation. Urinary back-flow from renal tubules can result in crystals appearing within the Bowman's space. It has been pointed out that some monotypic light chains reabsorbed by tubular cells were mainly V region fragments not easily recognized by commercially available antibodies (8). Demonstration of light chain restriction can be made more sensitive through the use of pronase-digested immunofluorescence staining, or immunohistochemistry by the antigen retrieval technique. In the series by Larsen et al, fewer LCPT cases were found to have crystals (1). This in contrast to the greater number of biopsies with crystals documented to date. The lack of sensitivity of frozen tissue immunofluorescence for detection of light chain restriction might have contributed to underreporting when crystals were not seen. On the other hand, one must be careful not to interpret higher  (21) but a report of the largest cohort of cases suggested that the overall survival is no different to non-IgM myelomas diagnosed and treated contemporaneously. However, it is clear that IgM MM definitely has a poorer overall outcome compared to LPL/WM. In addition, IgM MM appears to have poor clinical outcome in the context of high-dose therapy (22). At the present time, treatment guidelines recom mend the same therapeutic approach for IgM, IgG and IgA myelomas.

Conclusion
In summary, we have illustrated the renal pathology and clinical characteristics of LCPT secondary to a rare form of MM. The renal biopsy can be the sentinel investigation that alerts clinicians to an underlying life-threatening illness.