Chronic Myelomonocytic Leukemia Patients With Lysozyme Nephropathy and Renal Infiltration Display Markers of Severe Disease

Introduction Chronic myelomonocytic leukemia (CMML) is a hematologic disorder that is an overlap syndrome between myelodysplastic syndromes and myeloproliferative neoplasms, and can be associated with autoimmune and inflammatory diseases. This study aimed to describe kidney involvement in patients with CMML, their treatments, and outcomes. Methods We conducted a French and American multicenter retrospective study in 15 centers, identifying patients with CMML with acute kidney injury (AKI), chronic kidney disease (CKD), and urine abnormalities. Results Sixteen patients (males, n = 14; median age 76.5 years [71.9–83]) developed a kidney disease 6 months [1.6–25.6] after the diagnosis of CMML. At the time of kidney disease diagnosis, median urinary protein-to-creatinine ratio was 2 g/g [1.25–3.4], and median serum creatinine was 2.26 mg/dl [1.46–2.68]. Fourteen patients (87.5%) underwent a kidney biopsy, and the 2 main pathological findings were lysozyme nephropathy (56%) and renal infiltration by the CMML (37.5%). Ten patients received a new treatment following the CMML-associated kidney injury. Among patients with monitored kidney function, and after a median follow-up of 15 months [9.9–34.9], 4 patients had CKD stage 3, 4 had CKD stage 4, 1 had an end-stage kidney disease. In our patient series, 2 patients evolved to an acute myeloid leukemia (AML), and 5 died. Compared with 116 CMML controls, patients who had a kidney involvement had a higher monocyte count (P < 0.001), had more CMML-1 (P = 0.005), were more susceptible to develop an AML (P = 0.02), and were more eligible to receive a specific hematologic treatment, with hydroxyurea, or hypomethylating agents (P < 0.001), but no survival difference was seen between the 2 groups (P = 0.6978). Conclusion In this cohort of patients with CMML with a kidney injury, the 2 most frequent renal complications were lysozyme-induced nephropathy and renal infiltration by the CMML. Kidney involvement should be closely monitored in patients with CMML.

Introduction: Chronic myelomonocytic leukemia (CMML) is a hematologic disorder that is an overlap syndrome between myelodysplastic syndromes and myeloproliferative neoplasms, and can be associated with autoimmune and inflammatory diseases.This study aimed to describe kidney involvement in patients with CMML, their treatments, and outcomes.
Methods: We conducted a French and American multicenter retrospective study in 15 centers, identifying patients with CMML with acute kidney injury (AKI), chronic kidney disease (CKD), and urine abnormalities.
Results: Sixteen patients (males, n ¼ 14; median age 76.5 years [71.9-83])developed a kidney disease 6 months [1.6-25.6]after the diagnosis of CMML.At the time of kidney disease diagnosis, median urinary protein-to-creatinine ratio was 2 g/g [1.25-3.4],and median serum creatinine was 2.26 mg/dl [1.46-2.68].Fourteen patients (87.5%) underwent a kidney biopsy, and the 2 main pathological findings were lysozyme nephropathy (56%) and renal infiltration by the CMML (37.5%).Ten patients received a new treatment following the CMML-associated kidney injury.Among patients with monitored kidney function, and after a median follow-up of 15 months [9.9-34.9], 4 patients had CKD stage 3, 4 had CKD stage 4, 1 had an endstage kidney disease.In our patient series, 2 patients evolved to an acute myeloid leukemia (AML), and 5 died.Compared with 116 CMML controls, patients who had a kidney involvement had a higher monocyte count (P < 0.001), had more CMML-1 (P ¼ 0.005), were more susceptible to develop an AML (P ¼ 0.02), and were more eligible to receive a specific hematologic treatment, with hydroxyurea, or hypomethylating agents (P < 0.001), but no survival difference was seen between the 2 groups (P ¼ 0.6978).

Conclusion:
In this cohort of patients with CMML with a kidney injury, the 2 most frequent renal complications were lysozyme-induced nephropathy and renal infiltration by the CMML.Kidney involvement should be closely monitored in patients with CMML.
Kidney Int Rep (2023) 8, 2733-2741; https://doi.org/10.1016/j.ekir.2023.09.005KEYWORDS: acute kidney injury; chronic myelomonocytic leukemia ª 2023 International Society of Nephrology.Published by Elsevier Inc.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).K idney diseases in patients with hematological ma- lignancies are common, with various etiologies. 1 The development of kidney disease significantly worsens the prognosis of these patients.In the past few years, the concept of monoclonal gammopathy of renal significance has allowed the better characterization of kidney diseases related to monoclonal gammopathies. 2evertheless, renal complications associated with myeloid neoplasms are much less described.
CMML is an overlap syndrome that has characteristics of a myelodysplastic syndrome, whereas other findings are more consistent with the diagnosis of myeloproliferative neoplasm. 3CMML is characterized by persistent monocytosis, hypercellular marrow with granuloblastic hyperplasia, variable degrees of dysplasia, and increased monocytic cells.The diagnosis, prognostic, and therapeut management of CMML have been modified by the identification of molecular abnormalities. 46][7] The incidence of AKI and CKD is particularly high in patients with CMML (34.9%, and 7.6%, respectively). 8The landscape of kidney disorders in CMML is based on single-case studies and small cohorts.0][11][12] Glomerular injury description remains elusive.Kidney injuries related to CMML were described in small cohorts, generally associated with myeloproliferative neoplasms. 13,14There is a higher incidence of AKI (34.9%) and CKD (7.6%) among patients with myeloproliferative form of CMML. 8Main findings revealed that tubulointerstitial compartment was the first kidney injury in a study of 8 CMML cases, with chronic tubulointerstitial nephropathy, and acute interstitial nephropathy. 13An interstitial infiltration can be seen, by T cells, or a massive infiltration by the CMML.Another histologic study of 4 patients showed glomerular injuries such as endothelial damage, mesangiolysis, double contours, and rare lesions of podocytopathy and thrombotic microangiopathy. 14evertheless, the size of these cohorts remains small.The objective of this work was to study the clinical and histological presentation of patients with renal impairment during CMML, as well as their therapeutic management in a multicenter cohort.Then, we compared the hematological phenotype of patients with a kidney injury occurring during CMML with CMML patients' controls without kidney disease.

Patient Selection
Patients with an established diagnosis of CMML and diagnosed with AKI, CKD, or urine abnormalities were retrospectively included.We collected data of patients from 3 different sources (Supplementary Figure S1).The first group of patients was recruited at the Brigham and Women's Hospital, and the Massachusetts General Hospital, at Boston, MA, USA where we screened-in all consecutive patients with a diagnosis of CMML with a kidney biopsy over the past 20 years.The second one includes patients with a renal injury related to a diagnosis of vasculitis associated with CMML, from French different databases such as "Groupe Francophone des Myélodysplasies," and "French Network of Dysimmune Disorders Associated with Hemopathies" groups. 15Finally, we reached out to various French scientific societies ("Société Nationale Française de Médecine Interne," and "Société Francophone de Néphrologie Dialyse et Transplantation") to do a retrospective collection of patient cases that fitted our inclusion criteria.We excluded every kidney disease previously known before the diagnosis of CMML, and which remained stable after the myeloid neoplasm diagnosis.As detailed in Supplementary Figure S1, the kidney diagnosis that we excluded were cases of diabetic nephropathy, membranous nephropathy associated with graft versus host disease, and hypertensive nephrosclerosis.Controls with CMML without any kidney injury were extracted from GFM prospective nationwide database.

CMML Definitions
Diagnoses criteria for CMML were based on the World Health Organization classification revised in 2016.CMML were classified as CMML-0 (for cases with <2% blasts in the blood, and <5% blasts in the bone marrow), CMML-1 (2 to 4% blasts in the blood, and/or 5% to 9% blasts in the bone marrow), and CMML-2 (5% to 19% blasts in the blood, and 10% to 19% blasts in the bone marrow).Cytogenetic analysis and molecular findings were collected when available in the center.

Definition of Kidney Diseases
We included adult patients (>18 years old) diagnosed with CMML, and a kidney disease less than 10 years apart.According to the National Kidney Foundation's Kidney Disease Outcome and Quality Initiative guidelines, CKD is defined as either a decreased glomerular filtration rate (<60 ml/min per 1.73 m 2 ), or markers of kidney damage: albuminuria with albumin-tocreatinine ratio $30mg/g, urine sediment abnormalities, electrolyte and other abnormalities due to tubular disorders, and abnormalities detected by histology among other things. 16Estimated glomerular filtration rate (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration formula. 17idney biopsies were locally reviewed in each pathology center.

Clinical Data
Clinical and biological data were retrospectively collected from the patients' charts.Clinical data included gender, age at the time of the hematological and renal diagnoses, history of hypertension, and diabetes mellitus.Extrarenal organ involvement was included if it was directly attributable to systemic manifestations of the CMML.Biological data, at the time of the hematological and renal diagnoses, included blood cell counts, serum creatinine, eGFR, urinary protein-to-creatinine ratio, and urinary sediment.Regarding underlying CMML, data collection included bone marrow cytology and cytogenetic analyses, CMML-specific prognostic scoring system, treatments, and the evolution to AML.The presence, and the type of somatic mutations by next-generation sequencing were recorded if available.At the time of the renal diagnosis, we collected antinuclear antibodies, rheumatoid factor, antineutrophil cytoplasmic antibody testing by immunofluorescence and/or enzyme-linked immunosorbent assay, serum albumin, C-reactive protein, C3/C4 levels, and the type of treatment.

Follow-Up
During follow-up, events such as dialysis, kidney transplantation, occurrence of AML, or patient death were collected.Evaluation of kidney function with proteinuria and serum creatinine was performed at the diagnosis of CMML, at the diagnosis of the kidney injury, and at last follow-up.

Statistics
The descriptive data of the patients were expressed in number (frequency) for the binary variables, and in median (interquartile range) for the continuous variables.Given the size of the numbers and the lack of a normal distribution of values, we used nonparametric tests.Comparisons of continuous variables between 2 groups were made using the Mann-Whitney test, and those of binary variables were made using the Chi-square test.A Kaplan-Meier method was used to estimate overall survival.The significance level was P < 0.05.

Ethics
This study was conducted according to the Declaration of Helsinki and fulfilled the recommendations of the French "Commission Nationale Informatique & Libertés" CNIL.According to its policy, this study was approved by the Institutional Review Board of the Brigham and Women's Hospital (protocol number 2019P003787), who validated a waiver of consent.

Kidney and Systemic Manifestations
Time between the hematologic and kidney disease was 6 months [1.6-25.6](Table 1).The diagnosis of CMML preceded kidney disease in 9 patients.The 2 diseases were diagnosed at the same time for 4 patients.For 1 patient, the CMML was diagnosed after kidney disease (2 missing data).One patient required hemodialysis at renal The first indication of the kidney biopsy was AKI associated to a tubular proteinuria (n ¼ 9; 64%), then nephrotic syndrome (n ¼ 3; 20%), rapidly progressive glomerulonephritis (n ¼ 1; 7%), and a progressive CKD (n ¼ 1; 7%).A preexisting CKD of unknown cause with an eGFR of 33 and 44 ml/min per 1.73 m 2 was present in 2 patients.Hypertension was present in 82% of the patients, whereas diabetes represented 18%.Few patients had systemic symptoms (n ¼ 4, 25%) with 2 organs preferentially incriminated: skin and lungs (alveolar hemorrhage and purpura).Four patients had a monoclonal immunoglobulin (3 IgG kappa and 1 IgG lambda) with a median peak of 3 g/l [1.6-9].Median Creactive protein was 56 mg/l [9.6-100].Auto-immunity was detected in 5 of the 10 tested patients (antinuclear antibodies n ¼ 2, antineutrophil cytoplasmic antibody with no specificity n ¼ 2, and rheumatoid factor n ¼ 2).One patient had a detectable cryoglobulinemia associated with a diagnosis of lysozyme nephropathy.Regarding the serum complement analyses, 25% had a low C3 and 12.5% a low C4.

Renal Diagnoses and Histological Features of
Patients With CMML Among 16 patients, 14 (87.5%)underwent a kidney biopsy.The 2 most frequent diagnoses were lysozyme nephropathy (n ¼ 9, 56%), and an interstitial infiltrate compatible with a CMML infiltration in the kidney (n ¼ 6, 37.5%) (Table 1).Among the 3 patients with a nephrotic syndrome, 1 had a minimal change disease, 1 had lysozyme nephropathy with nephrotic range lysozymuria, and 1 had a renal CMML infiltrate with extramedullary hematopoiesis.One patient had both diagnoses.The features of light microscopy, immunofluorescence, and immunohistochemistry analyses of the kidney biopsies are listed in Supplementary Table S1.
Histological findings for patients with a diagnosis of lysozyme nephropathy showed lesions of acute tubular injury with focal tubular epithelial necrosis (n ¼ 4), and a vacuolization of the epithelial cells of the proximal tubules (n ¼ 7).Lysozyme staining was positive in 5 (n ¼ 62.5%) patients (4 immunohistochemistry and 1 immunofluorescence).Four patients with lysozyme nephropathy had also lysozymuria.Interstitial fibrosis ranged from 10% to 30%.Inflammatory infiltrate with mostly mononuclear myeloid cells was described in 9 patients.For the patients who had a characterization of the infiltrate by immunohistochemistry, MPO and CD68 staining were positive (Supplementary Table S1).Regarding the 2 patients without a kidney biopsy, 1 had a diagnosis of systemic vasculitis based on the skin biopsy showing C3 deposits along the capillary of the superficial derma, with a renal presentation associating AKI and glomerular proteinuria.The other patient had polyarteritis nodosa with renal microaneurysms.

Outcomes
At renal diagnosis, 3 patients were already treated for their CMML by hydroxyurea (Table 2).After the renal diagnosis, hematologic treatment was started or modified in 6 patients (37.5%).It consisted of the introduction of 1 or several of the following treatments: hydroxyurea, azacitidine, mercaptopurine, and an allogeneic stem cell transplantation.Three patients had a diagnosis of CMML concomitant to or less than 2 months after renal involvement.Each one received a different hematologic treatment: hydroxyurea, azacytidine, and decitabine.Four patients received steroids after the renal diagnosis.One of them was diagnosed with polyarteritis nodosa and his treatment consisted of cyclophosphamide, azathioprine, and steroids.

Comparison Between CMML With and Without Kidney Injuries
Compared to 116 patients with CMML without a kidney injury, patients with a CMML associated with a kidney involvement had a higher monocyte count (1.8 G/l [1.3-2.8] vs. 5 G/l [3.6-10.8],respectively, P < 0.001) (Table 3).Moreover, the main subtype of CMML in the group with a kidney injury was the CMML-1 (66.7% vs. 25% in the control group, P ¼ 0.005).Even if there was no difference in terms of prognostics, with an International Prognosis Scoring System-Revised above 3.5 in 28.4% of CMML control group against 20% (P ¼ 1), patients with CMML with a kidney injury were more susceptible to develop an AML: 30.8% versus 6.9% in the control group (P ¼ 0.02).Patients with CMML who had a kidney injury were more eligible to receive a specific hematologic treatment, with hydroxyurea, or hypomethylating agents (84.6% and 19.8% in the control CMML group, P < 0.001).The median followup from CMML diagnosis was 21 months [7-39.5] in patients without a kidney injury, and 34.9 months [13.9-46.5] in patients with a kidney injury (P ¼ 0.174).
The median survival was 59 months, and not reached for the follow-up period in CMML with and without a kidney injury, respectively (P ¼ 0.6978, odds ratio: 1.18 [95% confidence interval 0.43-3.28])(Figure 1).

DISCUSSION
CMML is a myeloid neoplasm characterized as a myelodysplastic syndrome/ myeloproliferative neoplasm overlap syndrome.CMML is associated with significant monocytosis, which is correlated with AKI and CKD. 8 In our study, the time between the diagnosis of CMML and the development of a kidney injury was 6 months, which is close to the delay reported by Grignano et al. 5 in their study describing autoimmune and inflammatory diseases associated with CMML.However, Belliere et al. 13 identified a delay of 7.7 AE 2 years for CMML or BCR-ABL-negative myeloproliferative neoplasms patients.We found that lysozyme nephropathy and an infiltration by the CMML were by far the most frequent kidney diseases associated with CMML.This was similar to the results of Gipe et al. 18 who described the spectrum of renal pathological findings among 14 patients with CMML.In large cohorts of autoimmune and inflammatory diseases associated with CMML, systemic vasculitis was the main related feature. 5,6Despite this fact, we onlyhad 2 cases of vasculitis in our cohort.This contradiction can be explained in part by the pathophysiology of the specific kidney injuries associated with CMML.
Lysozyme is a small cationic protein produced by monocytes, that is freely filtered by the glomerular filtration barrier, and reabsorbed by proximal tubules. 12An overproduction of lysozyme can be observed in CMML.Above a certain threshold, lysozyme becomes toxic to proximal tubular cells, leading to acute tubular injury.A kidney biopsy is needed to establish the diagnosis of lysozyme nephropathy, because elevated serum, and urine lysozyme levels commonly occur in patients with CMML, without being associated with the development of AKI. 19ysozyme-induced nephropathy is characterized by acute tubular injury with abundant cytoplasmic granular inclusions that stain strongly for lysozyme. 12mportantly, lysozyme-induced nephropathy can respond to renal supportive care and cytorectuction, arguing for a monitoring of lysozymuria in patients with CMML, and if necessary a renal biopsy to confirm the diagnosis. 18idney infiltration by CMML is mostly seen in the tubulointerstitial compartment, even if it has already been described in the glomerulus. 13,20,21Two types of infiltrates have been characterized by immunohistochemistry: extra-medullary hematopoiesis, with the presence of megakaryocytes (positive CD61 marker); and a myelomonocytic infiltrate (positive MPO and CD68 markers). 13,20However, it can be difficult to properly assess the percentage of renal parenchymal infiltration by myelomonocytic cells, because the nature of the infiltration can be patchy with a lot of inflammation.Further work assessing why the kidney is a target of the myelomonocytic infiltration are also needed.It has been shown that the presence of clonal hematopoiesis in bone marrow was also present in cutaneous immature myeloid cell infiltrate in a new entity called myelodysplasia cutis, because the same mutations were found in both tissues. 22This suggests that clonal monocytes could also participate in kidney lesions, with different hypothetical mechanisms involved such as chemokine gradient, kidney as a proliferation niche.
At last follow-up, kidney function evolution was heterogenous, with 4 patients evolving to CKD 3, 4 patients to CKD 4, and 1 patient to end-stage kidney disease, which was comparable to Belliere et al. 13 Interestingly, in Gipe et al. 8 study, although supportive care and cytoreductive therapy did help improve renal function in all patients with lysozymeinduced nephropathy, they continued to have CKD, potentially arguing for earlier cytoreduction and renal risk factor mitigation.Compared to CMML controls, patients with CMML who had a kidney injury were more eligible to receive a specific hematologic treatment.CMML associated with a kidney involvement seemed to have worse baseline prognostic factors (monocytes count, type of CMML, number of dysplasia, and evolution toward AML), but we did not observe a statistically significant difference in terms of overall survival.
Limitations of this work are mainly due to its retrospective design, and the small sample size of the cohort.To push further the analysis, the next objectives will be to collect kidney biopsy samples to further characterize the pathological features of the kidney injuries (infiltrate and lysozyme staining) associated with CMML.One of our perspectives will be to realize next-generation sequencing on the kidney biopsies of patients with an infiltrate, to see if we can find a significant renal clonal infiltrate of CMML cells.In conclusion, a regular screening for blood pressure, proteinuria, and kidney function should be proposed to all patients with CMML.In case of kidney dysfunction, kidney biopsy seems important to confirm the diagnosis in patients with CMML or to rule out differential diagnoses.Dosage of lysozymuria can also be informative, although not easily accessible in routine.In this cohort of patients with CMML with a kidney injury, the 2 most frequent kidney complications were lysozyme-induced nephropathy, and kidney infiltration by the CMML.The occurrence of a kidney injury in the course of a CMML seems to worsen the prognosis of these patients.

Figure 1 .
Figure 1.Kaplan Meier curve for the overall survival according to CMML with and without kidney injury.CMML, chronic myelomonocytic leukemia.

Table 3 .
Characteristics of CMML according to the presence/absence of a kidney injury AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; IPSS-R, International Prognosis Scoring System-Revised.a Missing data.