The Pathological Features of Leukaemic Cells Inltrating Renal Interstitium in Chronic Lymphocytic Leukaemia/small Lymphocytic Lymphoma Within 10 Years in a Large Single Chinese Centre

Background Chronic lymphocytic leukemia/Small Lymphocytic Lymphoma (CLL/SLL) was rare in Asians and patients with CLL/SLL rarely undergo kidney biopsy. Little information is available on the histopathological features and clinical relevance of CLL/SLL tubulointerstitial injury. Hence, we attempted to describe the clinical characteristics, renal pathology and clinical outcome of a well characterized population of CLL/SLL patients with CLL inltration in renal interstitium from a large single center in China. Methods Between January 1st, 2010 to September 31st, 2020, 31946 reanl biopsy pathology were performed at Peking University First Hospital and 10 patients with CLL inltration in renal interstitium were included. Complete clinical data were collected from these 10 patients and the renal specimens were examined by routine light microscopy, immunonuorescence and electron microscopy examination. Results The extent of inltrating CLL cells was diverse between different patients, from 10% to 90%. Five patients presented interstitial inltration with the percent of inltrate CLL cells ≥ 50%. Proliferation centers were formed in the renal interstitium of 6 patients (6/10, 60%). Intrestingly, we found three patients (3/10, 30%) expressed monoclonal immunoglobulin in inltrating CLL cells and special crystal structures were found in these cells in two of the three patients for the rst time. Conclusions The nding conrmed CLL cells inltrating in renal interstitium can secret monoclonal immunoglobulins directly, indicating that CLL cells in renal interstitium probably directly involved in local lesion injurys by secrecting monoclonal immunoglobulin. This nding will help understanding the mechanism of renal injury with CLL inltration, thereby improving our current therapeutic efforts.


Background
Chronic lymphocytic leukemia/Small Lymphocytic Lymphoma (CLL/SLL), the most common adult leukemia in the Western world [1], however, infrequent in Asians. It is characterized by the clonal expansion of CD5 + CD23 + B cells in blood, marrow, and secondary lymphoid tissues. Extramedullary/Extranodal manifestations of CLL/SLL are rare. Most often involved organs are the skin and central nervous system. Kidneys were rarely involved [2][3]. A study from the Mayo Clinic found a 7.5% incidence of renal insu ciency at the time of CLL/SLL diagnosis in a cohort of over 4000 CLL/SLL patients. Importantly, the presence of kidney disease is independently associated with adverse patient outcomes in CLL/SLL [4], which emphasized the importance of detailed exploration of the mechanism of renal insu ciency in CLL/SLL.
In renal complications of CLL/SLL, the mechanism of renal insu ciency in such patients varies. Besides glomerular injury which including direct glomerular deposition of monoclonal protein, cryoglobulins, and immune-complexes [4][5], renal tubulointerstitial injury was also an important mechanism. Previous case reports and studies mainly focused on glomerular injury associated with CLL/SLL. However, little information is available on the histopathological features and clinical relevance of CLL/SLL tubulointerstitial injury. Up to date, literatures related to CLL in ltration of the kidney were either single patient case reports or small case series [4][5][6][7][8][9][10], with the largest report including 6 patients [4]. In these literatures, histopathological features of CLL cells in ltrating in renal interstitium were not detailed described and the mechanism of renal injury with CLL in ltration was not studied.
In this study, we attempted to describe the clinical characteristics, renal pathology and clinical outcome of a well characterized population of CLL/SLL patients with CLL in ltration in renal interstitium from a large single centre at Peking University First Hospital in China. We detailed explored histopathological changes of the tubular and interstitium and the pathogenesis of CLL cells in ltrating in renal interstitium. light chain, lambda light chain and IgG subclasses. Electron microscopy was performed as per clinical routine. Immunohistochemical staining for CD3, CD5, CD20, CD23, Cyclin D1, CD43, TdT, CD10, BCL6 were done on renal biopsy to assess CLL/SLL involvement. Two pathologists specializing in evaluation of renal pathology evaluated renal biopsies separately. All cases were also reviewed by hematopathologists specializing in review of lymphoid malignancy.
Differences in diagnosis between the two pathologists were resolved by re-reviewing the biopsies to reach a consensus.

Immunoelectron microscopy (IEM)
Immuno-gold labeling was further performed according to the methods described by previous studies [12]. The 80-nm-thick resin-embedded sections were attached on the surface of a carbon-coated support lm grid and blocked with 2% bovine serum albumin in 0.01 M phosphate buffer saline, pH7.4, saline for 5 min at 37℃, followed by incubation overnight at 4 ℃, using an extensive panel of antibodies reactive with polyclonal rabbit anti-human kappa and lambda light chain (1:1000; Dako, Carpenteria, CA), monoclonal mouse anti-human IgG1, IgG2, IgG3, IgG4 (1:100; SouthernBiotech, Birmingham, AL). Then, after incubation with a gold-conjugated secondary antibody (colloid gold particle in a diameter of 10 nm) for 1 h, the grid was ready for observation using the transmission electron microscope. Negative controls were prepared by omitting the primary antibody.

Statistical analysis
Statistical software SPSS 13.0 (SPSS, Chicago, IL, USA) was used. Descriptive statistics of baseline characteristics were calculated. Continuous data were expressed as mean ± s.d, or median with range. Categorical variables were presented as proportions.

Demographic and baseline clinical data
The clinical features at biopsy are presented in Table 1. 5 male (50%) and 5 female (50%) were enrolled. The mean age was 64±4.9 (range 58-73) years old.
The CLL/SLL had been diagnosed before kidney biopsy in 5/10 cases, with a mean interval of 23.8 months (range 2-60months). Four patients were biopsied for acute kidney disease, two for acute kidney injury and four for proteinuria. The mean peak serum creatinine before renal biopsy was 276.6±192.3μmol/L (range: 95-616μmol/L). The median proteinuria was 3.5g/24hr (range: 0.48-12.8) with a mean serum albumin of 33.6±9.1g/L. Seven patients (70%) had microscopy hematuria. All patients had bone marrow biopsy, and eight patients (80%) showed CLL/SLL bone marrow in ltration. Nine patients (90%) had multiple enlarged lymph nodes, and all of them had lymph node biopsy with the de nition of SLL. One patients had splenomegaly. Six patients (60%) had monoclonal immunoglobulins, including 3 patients with IgMκ, 2 patients with IgGκ and 1 patient with IgMλ.

Renal biopsy ndings
Tables 2 summarize the pathology characteristics of the kidney biopsy. All of these patients presented varying degrees of CLL cells in ltration in renal interstitium, composed mainly of small mature lymphocytes, with small hyperchromatic nuclei, expanding the interstitium at the expense of the tubular structures, the peritubular capillaries, and less frequently the glomeruli ( Figure 1). Immunohistochemistry revealed monotypic lymphocytes (CD20+, CD5+, CD23+, CyclinD1-, CD10-, CD138-, CD68-) ( Figure 1). The extent of in ltrating CLL cells was diverse between different patients, from 10% to 90%. Five patients presented interstitial in ltration with the percent of in ltrate CLL cells≥50%.
Proliferation centers were found in 6 patients (60%), which are concentrations of prolymphocytes and paraimmunoblasts. This was a spectrum of larger cells with more dispersed chromatin, conspicuous nucleoli, and more abundant cytoplasm than small mature lymphocytes ( Figure 1). Of the 6 patients, two patients (Case 6 and Case10) showed monoclonal immunoglobulin expressions in renal interstitium by immuno uorescence (IgG1κ for case6 and IgMκ for case10), which were identical to that of the serum immunoglobulin paraprotein. Interestingly, special crystal structures were found in in ltrating CLL cells in one patient (Case 6) and fuzzy lament structure can be seen in these crystal structures ( Figure 2). The special structures formed in in ltrating CLL cells was different from those formed in glomerulus which were characterized by 40-nm diameter microtubules ( Figure 2). Notably, IgG1κ expression was detected in these crystals by immunoelectron microscopy (Figure 3). In another patient (Case 2) whose immuno uorescence showed no speci c interstitial deposit, rhombic crystal formation was also found in in ltrating CLL cells and monoclonal λ was expressed in these cystals by immunoelectron microscopy (Figure 4).
Totally, three patients (30%) expressed monoclonal immunoglobulin in in ltrating CLL cells. In addition, non-necrotic epithelioid granulomas were found in one patient (Case 5). Light microscopy revealed interstitial gigantocellular granulomas, surrounded by lymphocytes ( Figure 1). Periodic Acid Schiff, Ziehl and Grocott stainings revealed no microorganism. Immuno uorescence showed no speci c interstitial deposit.

Treatment and prognosis
Nine patients had follow-up information. The mean post-biopsy follow-up was 26.1 months (range 2-56 months). As shown in table 1, four patients received chemotherapy. One patient (Case 5) was treated with 2 cycles of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) before kidney biopsy, and after the renal biopsy, she was treated with ibrutinib. Her renal function achieved partial recovery and CLL/SLL showed no sign of progression. Two patients (Case3 and 6) were treated with chemotherapy after kidney biopsy, including cyclophosphamide, vincristine, and prednisone(COP) followed by rituximab and ibrutinib, and their CLL/SLL were stable. One patient (Case 4) was treated with 1 cycle of rituximab, udarabine and cyclophosphamide (RFC) and discontinued due to patient's reluctance of chemotherapy, and her renal function improved with CLL/SLL stable for a 31 months follow up time. Five patients don't receive chemotherapy. one patients (Case 1) was treated with interferon for 2 months and discontinued after kidney biopsy. Then he was treated with cyclophosphamide and cyclosporine for membranous nephropathy but with no remission. Patient 7 was only treated with prednisone and her renal function improved. Patient 8 was treated with plasma exchange for thrombotic microangiopathy which showed hematologic complete remission and recovery of acute kidney injury for 2 episodes. Chemotherapy was suggested for patient 2 and 9 but they refused.

Discussion
CLL/SLL is extremely rare in Asians compared with persons of predominately European descent [1]. Unlike plasma cell dyscrasias, in which kidney biopsy is routinely performed to evaluate unexplained renal insu ciency [13], patients with CLL/SLL rarely undergo kidney biopsy due to the fact that CLL/SLL usually follows an indolent course. In the series from Mayo Clinic [4], of all the CLL/SLL patients studied, only 1.2% of patients underwent kidney biopsy. The low rate of kidney biopsy is a limiting factor in our understanding of CLL/SLL associated kidney disease. Here, we attempted to better describe the pathological features of CLL/SLL patients with CLL in ltration in renal interstitium in Chinese patients. Particularly, we also showed CLL cells in renal interstitium expressed monoclonal immunoglobulin with crystals formation for the rst time. This pathological feature might provide useful information in the understanding of the mechanism of renal injury with CLL cells in ltration.
In CLL/SLL, the prevalence rates of hypogammaglobulinemia and monoclonal paraprotein in serum are 25% and 11% [14][15], respectively. The presence of serum paraprotein has been showed a worse prognosis of CLL/SLL patients [16][17]. In our study, 30% of patients expressed monoclonal immunoglobulin in CLL cells in ltrating in renal interstitium, which indicating CLL cells probably directly involved in local lesion injurys by secrecting monoclonal protein. Up to date, the mechanism of renal injury with CLL in ltration is not clearly established but has been hypothesized to involve tubular/microvascular compression causing intrarenal obstruction in addition to an in ltration-associated in ammatory/cytokine response [3][4][5]. For example, diffuse in ltration likely compresses the renal tubules and microvasculature, resulting in intrarenal obstruction and ischemia. Alternatively, cytokines such as IL-1, IL-6, TNF-α, and TGF-β, secreted directly by the lymphoma cells, may lead to tubular injury and interstitial brosis [18]. Our study indicated that the CLL cells in ltrating in kidney interstitium directly secreted the monoclonal protein. Because the presence of excess free-light chains with crystals formation was su cient to cause injury to renal proximal tubular cells such as light chain proximal tubulopathy (LCPT) [19], the injury of monoclonal immunoglobulin secreted by CLL cells in kidney interstitium should be assessed.

The occurrence of secreting CLL cells is related either to the development of CLL cells in situ or to the in ltration from CLL cells in the blood. Due to CLL cells
in the blood appear to be predominantly resting lymphocytes in G0 [20], focal aggregates of different-sized lymphocytes in the local lymphoid tissue are presumably where leukemia cells proliferate. CLL cell proliferation occurs in local sites termed proliferation centers or pseudofollicles [21]. Interactions between CLL and accessory cells within proliferation centers are critical for providing growth and survival signals to CLL B cells, inducing their proliferation, differentiate either to an antibody-secreting cell or a memory cell, or to remain a nonsecreting blast [22]. In our study, proliferation centers were found in 60% of patients with CLL in ltration in kidney interstitium, which indicated the kidney microenvironment plays a central role in the pathogenesis of CLL. Growing evidence suggests that different types of stromal cells, such as monocyte-derived nurselike cells, mesenchymal stromal cells and follicular dendritic cells protect CLL cells in coculture and are an integral part of the CLL microenvironment [21][22][23][24][25] Recently, pathways deliver critical survival and drug-resistance signals in the interactions that occur between CLL cells and their microenvironments has gained increasing attention in hemato-oncology, such as CD44, CXCL12,CXCL13,TNF family member BAFF [23,[26][27][28], and et al. Our study indicated that kidney was most likely another site of minimal residual disease and the source of relapses in patients with CLL. More therapeutic efforts may be put on the inhibition of CLL cell proliferation in kidney interstitium in further, because the presence of excess monoclonal protein with crystals formation was su cient to cause injury to renal tissues.
This study had some limitations. First, this was a retrospective study. Because more attention were paid to glomeruli in earlier years, we can not exclued the possibility that monoclonal immunoglobulin may be missed when interpretating the immuno uorescence stains in kidney interstitium. Second, the clinicopathological relationship of proliferation center in kidney interstitium or even monoclonal immunoglobulin expression in CLL cells is still unclear due to the limited sample size. A larger number of multicenter research will be needed in the further.

Conclusions
In summary, we attempted to better describe the pathological features of CLL/SLL patients with CLL in ltration in renal interstitium in Chinese patients. It is noteworthy that monoclonal immunoglobulin expression were dectected in CLL cells in ltrating renal interstitium for the rst time. The nding con rmed CLL cells in renal interstitium can secret monoclonal immunoglobulins directly, indicating these cells probably directly involved in local lesion injurys by secrecting monoclonal immunoglobulin. This will help understanding the mechanism of renal injury with CLL in ltration, thereby improving our current therapeutic efforts. Ethics approval and consent to participate

Abbreviations
The research was in compliance with the declaration of Helsinki and approved by the ethic committee of Peking University First Hospital.

Informed consent statement
All patients have signed an informed consent form on inclusion and at each follow-up assessment.

Consent for publication
Written informed consent was obtained from the patient for publication of the case report and any accompanying images.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
All authors declare no con ict of interest.   Representative electron microscopy ndings of renal biopsy in case 6. A. Large subendothelial deposits characterized by microtubule formation were detected in glomeruli (×15000). B. Enlargement of picture A showed 40-nm diameter microtubule formation as indicated by arrows (×50000). C.Neoplastic cells in ltration was seen in renal interstitium (×10000). D. Special crystal structures characterized by fuzzy lament structure were found in in ltrating neoplastic cells as indicated by arrows (×40000).

Figure 3
Representative immunohistochemistry ndings of renal biopsy in case 6. A. Scattered granular staining of IgG1 was shown in the interstitium as indicated by the arrow with crumby staining in glomeruli indicated by the arrowhead (Immuno uorescence staining, ×200). B. Scattered granular staining of C3 was shown in the interstitium as indicated by the arrow with crumby staining in glomeruli indicated by the arrowhead (Immuno uorescence staining, ×200). C. Intense IgG1 was detected in the crystal structures of neoplastic cells as indicated by the arrow. (Immunoelectron microscopy labelling, ×30000). D. Intense C3 was detected in the crystal structures of neoplastic cells as indicated by the arrow. (Immunoelectron microscopy labelling, ×25000).

Figure 4
Representative electron microscopy ndings of renal biopsy in case 2. A. Neoplastic cells in ltration was seen in renal interstitium (×8000). B. Rhombic crystals were formed in in ltrating neoplastic cells as indicated the arrow (×25000). C. Light kappa was not detected in neoplastic cells. (Immunoelectron microscopy labelling, ×30000). D. Intense light lamda was detected in the crystal structures of neoplastic cells as indicated by the arrow. (Immunoelectron microscopy labelling, ×20000).