Toskic, Denis

ABSTRACT: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2b randomized placebo-controlled, clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. Before drug administration, renal biopsies were taken and analyzed. OBJECTIVE: Identifying amyloid-specific molecular signatures in kidney biopsy tissue. MATERIALS & METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Clinical data was collected and correlated to histologic and molecular findings. Histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial (TI) compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined . RESULTS: Ten patients were enrolled, randomized and treated in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and TI compartments ( See Figure 1). Key clinical data pertaining to kidney function and biopsy morphology by compartment and cluster type were documented. Despite similar eGFR and degree of proteinuria, there was a significantly higher AS score in the TI compartment (6.5 vs. 4.5; p=0.0290) of G2 which was driven most entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI compartment. Other enriched ingenuity canonical pathways to note in the TI included ‘coagulation system’, ‘GADD45 signaling’, ‘Wnt/Ca+ pathway’, ‘intrinsic prothrombin activation pathway’ among others. Of note, genes like BRCA and CDKN1A were enriched between G1 and G2, both of which have been associated with molecular mechanisms that underly certain neoplasms ( See Figure 2). Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli, there was increased signaling of TNF, NFKB, IL33, IL-8 and IL15, consistent with an important inflammatory response. There was also an enrichment in pathways related to migration, adhesion and integrin signaling. PI3K/Akt signaling was also noted. Tubules showed increased activation of transcriptional regulators TP53, TP73 and PI3K/AKT signaling. Some gene regulators of cellular proliferation such as ABL1, BCL2 and AKT1 were found to be enriched in the amyloid group. CONCLUSIONS: While the RAIN trial closed prematurely and NEOD001 cannot be evaluated, the molecular data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in of themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Based on this early work, targeting the PI3K/Akt pathway may be a worthy pursuit in this disease.

Background: Seventy percent of patients with light chain (AL) amyloidosis present with renal involvement. The phases of the disease include asymptomatic seeding, increasing proteinuria, and progression to end stage renal disease. In an attempt to identify genes relevant to renal reprogramming due to persistent disease,renal biopsies from treated AL patients were obtained for systematic histologic evaluation and transcriptional profiling. Correlates were sought between these two approaches. Methods: Renal biopsies from 10 patients with persistent proteinuria (>500mg/day) following hematologic responses to anti-plasma cell therapy were scored independently by 2 expert renal pathologists employing a novel histologic tool that assessed scarring, fibrosis and the distribution of amyloid, and were successfully evaluated by transcriptional profiling in the Michigan Kidney Translational Medicine Core lab. Clinical data and histologic scores (CSIC = composite scarring injury score, AS = amyloid score) were correlated to the expression profiles of specific tubular and glomerular gene sets. Results: Baseline characteristics and histologic scores are in Table 1. Using a false discovery rate corrected P-value of < 0.10, numerous genes of interest were identified (Table 2). Transcriptional profiling revealed 2 distinct patient clusters (G1 and G2) within the tubular and glomerular gene expression sets. The histopathologic features were different between G1 and G2; the amyloid score was significantly higher in G2 in both the tubular (7.0 vs. 4.25; p=0.03) and the glomerular (6.92 vs. 4.38; p= 0.04) compartments. The differential expression for the genes of interest between G1 and G2 were also determined (Table 2). Conclusion: This effort has generated multiple genes of interest. ICQD and PODXL, in particular, correlated with the distribution of amyloid in the capillaries and mesangium. We are currently comparing these expression profiles to those of other nephropathies to identify genes whose expression levels may be specific to AL nephropathy, hopefully leading to further novel histologic and protein-based investigations. Disclosures Comenzo: Takeda: Research Funding; Prothena Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Caelum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Unum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Myself: Patents & Royalties: Patent 9593332, Pending 20170008966.