Delayed identification of monoclonal protein is associated with early death in isolated cardiac AL amyloidosis.

BACKGROUND: Early identification of immunoglobulin light-chain amyloidosis (AL) is crucial due to its rapid progression. Monoclonal light-chain (M-LC) testing is the first step in the diagnostic workup for patients with suspected cardiac amyloidosis (CA). We aimed to determine whether the time interval between the first CA suspicion and M-LC testing can be related to AL amyloidosis survival outcomes. METHODS: All patients (n = 94) with isolated cardiac AL amyloidosis diagnosed at our center between 2016 and 2020 were included. Those with pre-existing known monoclonal protein (monoclonal gammopathy of undetermined significance or smoldering multiple myeloma) were excluded. Time intervals to diagnostic tests and diagnosis were calculated and assessed for their survival prediction ability. RESULTS: The time interval between first CA suspicion (on echocardiography) and M-LC testing correlated with early mortality, and the best cutoff predicting survival, was 6 weeks. The 26 patients (∼28% of entire cohort) who underwent M-LC-studies >6 weeks after first suspicion more frequently presented Mayo stage IIIb (65% vs. 35%, p = .008), showing poorer overall survival than those (n = 68, 72%) referred for early M-LC studies (median 3 vs. 14 months, p = .039). CONCLUSIONS: Monoclonal protein testing should be the first-step in the diagnostic workup for patients with echocardiographic/other instrumental red flags raising CA suspicion.

A real-life study of daratumumab combinations in newly diagnosed patients with light chain (AL) amyloidosis.

Daratumumab-based regimens are the new standard of care for newly diagnosed patients with AL amyloidosis based on the results of the ANDROMEDA study. However, real-world data on daratumumab efficacy in upfront therapy in unselected patients are scanty. In the framework of a prospective observational study, we investigated the efficacy and safety of daratumumab in 88 newly diagnosed patients, including subjects with IIIb cardiac stage (26%) or myeloma defining events (29%). Daratumumab was administered with bortezomib in 50 (56%) patients, lenalidomide in 31 (35%), and monotherapy in 7 (8%). The rate of serious adverse events was low (16%). The overall hematologic response rate was 75% with 52 (59%) patients attaining at least a very good partial response (VGPR) at six months. Amongst patients evaluable for organ response, the rate of cardiac and renal responses at 6 months was 31% and 21%, respectively. Comparing stage IIIb patients with the remaining ones, the rate of profound hematologic response was not significantly different (≥VGPR 57% vs. 59%, p 0.955) likewise the rate of cardiac (33% vs. 30%, p 0.340) and renal (40% vs. 16%, p 0.908) responses. Daratumumab-based regimens demonstrated to be safe and effective in treatment-naïve AL amyloidosis even in advanced stage disease.

A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets.

In the next-generation sequencing era, RT-qPCR is still widely employed to quantify levels of nucleic acids of interest due to its popularity, versatility, and limited costs. The measurement of transcriptional levels through RT-qPCR critically depends on reference genes used for normalization. Here, we devised a strategy to select appropriate reference genes for a specific clinical/experimental setting based on publicly available transcriptomic datasets and a pipeline for RT-qPCR assay design and validation. As a proof-of-principle, we applied this strategy to identify and validate reference genes for transcriptional studies of bone-marrow plasma cells from patients with AL amyloidosis. We performed a systematic review of published literature to compile a list of 163 candidate reference genes for RT-qPCR experiments employing human samples. Next, we interrogated the Gene Expression Omnibus to assess expression levels of these genes in published transcriptomic studies on bone-marrow plasma cells from patients with different plasma cell dyscrasias and identified the most stably expressed genes as candidate normalizing genes. Experimental validation on bone-marrow plasma cells showed the superiority of candidate reference genes identified through this strategy over commonly employed "housekeeping" genes. The strategy presented here may apply to other clinical and experimental settings for which publicly available transcriptomic datasets are available.

Early cardiac response is possible in stage IIIb cardiac AL amyloidosis and is associated with prolonged survival.

Patients with immunoglobulin light chain (AL) amyloidosis and stage IIIb cardiac involvement have a dismal outcome despite the introduction of novel treatments. However, a rapid hematologic response translates in better survival. We evaluated the impact of early cardiac response and its depth on outcome in 249 patients with newly diagnosed stage IIIb cardiac AL amyloidosis. Hematologic and cardiac responses were evaluated by intent to treat. After a median follow-up of 52 months, 219 (84%) patients died, and median survival was 4.2 months. The 30- and 90-day hematologic response rates were 22% (at least very good partial response [VGPR] in 9%) and 24% (at least VGPR in 15%), respectively. Early hematologic response resulted in better survival. At 90 days, 21 (8%) patients achieved a cardiac response (cardiac very good partial response [cardiac VGPR] in 12 cases and cardiac partial response [cardiac PR] in 9). At the 90-day landmark analysis, cardiac response resulted in longer survival (median, 54 months), also in those patients who have achieved at least VGPR (median, 62 vs 26 months, P = .011). Patients with cardiac VGPR had a longer survival than those with cardiac PR (median, 92 vs 24 months; P = .027), whereas patients without cardiac response had a poor survival (median, 6 months). A baseline difference of involved/uninvolved free light chains > 50 mg/L (odds ratio [OR], 0.21, P = .024) and a bone marrow plasma cell infiltrate > 10% (OR, 0.23, P = .040) were negative predictors of 90-day cardiac response. Early cardiac responses are rare but possible in stage IIIb AL amyloidosis and translate to longer survival.

An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis.

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.

Prospective urinary albumin/creatinine ratio for diagnosis, staging, and organ response assessment in renal AL amyloidosis: results from a large cohort of patients.

OBJECTIVES: Quantification of 24 h-proteinuria is the gold standard for diagnosing, staging, and monitoring of patients with renal AL amyloidosis. However, 24 h-urine collection is cumbersome and may result in preanalytical error. In this prospective study, we investigated the role of urinary albumin/creatinine ratio (UACR) (cut-off: 300 mg/g) identifying renal involvement, evaluated a UACR-based staging system (UACR cut-off: 3,600 mg/g) and assessed whether UACR response (UACR decrease >30% without worsening in eGFR >25%) predicts renal outcome in 531 patients with newly-diagnosed AL amyloidosis. METHODS: From October 2013 paired 24 h-proteinuria and UACR (on first morning void) were measured in all newly-diagnosed patients with AL amyloidosis. Correlation between 24 h-proteinuria and UACR at baseline was assessed by Pearson's r test. Impact of UACR response on renal outcome was assessed in randomly created testing (n=354) and validation (n=177) cohorts. RESULTS: A strong linear correlation was found between 24 h-proteinuria and UACR at baseline (r=0.90; p<0.001). After a median follow-up of 31 months, 57 (11%) patients required dialysis. A UACR-based renal staging system identified three stages with significantly higher dialysis rate at 36 months comparing stage I with stage II and stage II with stage III. Achieving a renal response, according to a UACR-based criterion, resulted in lower dialysis rate in both testing and validation cohorts. CONCLUSIONS: UACR is a reliable marker for diagnosis, prognosis, and organ response assessment in renal AL amyloidosis and can reliably replace 24 h-proteinuria in clinical trials and individual patients' management.

Protease-sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation.

Light-chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL λ amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in 'proteolysis-prone' regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients.

Lenalidomide and dexamethasone in relapsed/refractory immunoglobulin light chain (AL) amyloidosis: results from a large cohort of patients with long follow-up.

Lenalidomide and dexamethasone (RD) is a standard treatment in relapsed/refractory immunoglobulin light chain (AL) amyloidosis (RRAL). We retrospectively investigated toxicity, efficacy and prognostic markers in 260 patients with RRAL. Patients received a median of two prior treatment lines (68% had been bortezomib-refractory; 33% had received high-dose melphalan). The median treatment duration was four cycles. The 3-month haematological response rate was 31% [very good haematological response (VGHR) in 18%]. The median follow-up was 56·5 months and the median overall survival (OS) and haematological event-free survival (haemEFS) were 32 and 9 months. The 2-year dialysis rate was 15%. VGHR resulted in better OS (62 vs. 26 months, P < 0·001). Cardiac progression predicted worse survival (22 vs. 40 months, P = 0·027), although N-terminal prohormone of brain natriuretic peptide (NT-proBNP) increase was frequently observed. Multivariable analysis identified these prognostic factors: NT-proBNP for OS [hazard ratio (HR) 1·71; P < 0·001]; gain 1q21 for haemEFS (HR 1·68, P = 0·014), with a trend for OS (HR 1·47, P = 0·084); difference between involved and uninvolved free light chains (dFLC) and light chain isotype for OS (HR 2·22, P < 0·001; HR 1·62, P = 0·016) and haemEFS (HR 1·88, P < 0·001; HR 1·59, P = 0·008). Estimated glomerular filtration rate (HR 0·71, P = 0·004) and 24-h proteinuria (HR 1·10, P = 0·004) were prognostic for renal survival. In conclusion, clonal and organ biomarkers at baseline identify patients with favourable outcome, while VGHR and cardiac progression define prognosis during RD treatment.

A safety review of drug treatments for patients with systemic immunoglobulin light chain (AL) amyloidosis.

INTRODUCTION: In AL amyloidosis, a usually small plasma cell clone secretes unstable, amyloid-forming light chains, causing cytotoxicity and progressive (multi)organ function deterioration. Treatment aims at reducing/eradicating the underlying clone, to reduce/zero the supply of the amyloidogenic protein and halt the amyloidogenic cascade. AREAS COVERED: Safety data of alkylating agents, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies from clinical trials are reviewed. EXPERT OPINION: Drugs used to treat AL amyloidosis are derived from experience with multiple myeloma or other B cell malignancies. However, treating AL amyloidosis is particularly challenging, as it implies delivering anti-neoplastic therapy to a hematologic malignancy directly causing (multi)organ function deterioration, often in elderly subjects with other comorbidities and polypharmacotherapy. This unique combination translates in increased patients' frailty and higher sensitivity toward treatment-related toxicities. Therefore, dose/schedule adjustments and special precautions are needed when translating treatment experience from multiple myeloma or other B cell malignancies to AL amyloidosis. Treatment of patients with AL amyloidosis should be risk adapted, tailored to individual patients' risk profile, considering the type and extent of organ involvement, and eventual comorbidity. As several classes of effective anti-plasma cell or B cell drugs are available, therapeutic choices are also influenced by individual drug's safety profile.

Minimal residual disease negativity by next-generation flow cytometry is associated with improved organ response in AL amyloidosis.

Light chain (AL) amyloidosis is caused by a small B-cell clone producing light chains that form amyloid deposits and cause organ dysfunction. Chemotherapy aims at suppressing the production of the toxic light chain (LC) and restore organ function. However, even complete hematologic response (CR), defined as negative serum and urine immunofixation and normalized free LC ratio, does not always translate into organ response. Next-generation flow (NGF) cytometry is used to detect minimal residual disease (MRD) in multiple myeloma. We evaluated MRD by NGF in 92 AL amyloidosis patients in CR. Fifty-four percent had persistent MRD (median 0.03% abnormal plasma cells). There were no differences in baseline clinical variables in patients with or without detectable MRD. Undetectable MRD was associated with higher rates of renal (90% vs 62%, p = 0.006) and cardiac response (95% vs 75%, p = 0.023). Hematologic progression was more frequent in MRD positive (0 vs 25% at 1 year, p = 0.001). Altogether, NGF can detect MRD in approximately half the AL amyloidosis patients in CR, and persistent MRD can explain persistent organ dysfunction. Thus, this study supports testing MRD in CR patients, especially if not accompanied by organ response. In case MRD persists, further treatment could be considered, carefully balancing residual organ damage, patient frailty, and possible toxicity.

Indicators of profound hematologic response in AL amyloidosis: complete response remains the goal of therapy.

In AL amyloidosis complete response (aCR) is defined as negative serum and urine immunofixation with normalized free light chain ratio (FLCR). However, achievement of low levels of involved FLC (iFLC) or difference between iFLC and uninvolved FLC (dFLC) are also relevant endpoints for treatment. We divided 434 consecutive patients with AL amyloidosis into five groups according to response 6 months after treatment initiation: aCR, iFLC <20 mg/L, normalized-iFLC, dFLC <10 mg/L, and normalized FLC ratio. Overall survival (OS) was similar (median not reached) in patients in aCR and in those who reached iFLC <20 mg/L, while it was inferior in all other groups (medians ranging from 79 to 91 months). Time to next therapy or death (TNTD) was longer in subjects attaining aCR (median 69 months) than in subjects reaching any FLC endpoint (medians ranging from 18 to 39 months). The ability of discriminating patients who survived more than 2 years among all responders was greater for current definition of aCR compared to combination of negative serum and urine immunofixation with any low-FLC endpoint. Complete response predicts best outcomes in AL amyloidosis and should be the goal of therapy if tolerability allows.

Sequential response-driven bortezomib-based therapy followed by autologous stem cell transplant in AL amyloidosis.

Autologous stem cell transplant (ASCT) is highly effective in selected patients with light chain (AL) amyloidosis. Bortezomib, preceding or following ASCT, improves responses. Satisfactory responses, including at least a partial response, very good partial response (VGPR) with organ response, or complete response, can be observed after induction therapy alone. We report 139 patients treated upfront with cyclophosphamide/bortezomib/dexamethasone (CyBorD), followed by ASCT only if response was unsatisfactory. Only 1 treatment-related death was observed. After CyBorD, hematologic response (HR) rate was 68% (VGPR or better, 51%), with 45% satisfactory responses. Transplant was performed in 55 (40%) subjects and resulted in an 80% HR rate (65% ≥ VGPR). Five-year survival was 86% and 84% in patients treated with ASCT or CyBorD alone, respectively (P = .438). Also, 6- and 12- month landmark analyses did not show differences in survival. Duration of response was not different in the 2 groups (60 vs 49 months; P = .670). Twenty-one (15%) patients with an unsatisfactory response to CyBorD could not undergo ASCT because of ineligibility or refusal; instead, they received rescue chemotherapy, with HR in 38% of cases and 51% 5-year survival. This sequential response-driven approach, offering ASCT to patients who do not attain satisfactory response to upfront CyBorD, is very safe and effective in AL amyloidosis.

Localized immunoglobulin light chain amyloidosis: Novel insights including prognostic factors for local progression.

In localized light chain amyloidosis (locAL), amyloidogenic light chains (aLC) are produced and deposited locally by a B-cell clone. We present 293 patients with immunohistochemically confirmed locAL. Lung (nodular pulmonary) with 63 patients was the most involved organ. The aLC was λ in 217 cases (κ:λ ratio 1:3). A local B-cell clone was identified in 30% of cases. Sixty-one (21%) had a concomitant autoimmune disorder (cAD). A monoclonal component (MC) were present in 101 (34%) patients and were more frequent in subjects with cAD (51% vs 34%; P = .03). Cigarette smoking was more prevalent in lung locAL (54% vs 37%; P = .018). After a median follow-up of 44 months, 16 patients died and 5- and 10-years locAL progression-free survival (PFS) were 62% and 44%. Interestingly, locAL-PFS was shorter among patients with an identified clonal infiltrate at amyloid deposition site (40 vs 109 months; P = .02) and multinuclear giant cells and/or an inflammatory infiltrate resulted in longer locAL-PFS in lung involvement (65 vs 42 months; P = .01). However, no differences in locAL PFS were observed in patients with cAD, a MC and involved organ site. Treatment was administered in 163 (54%) patients and was surgical in 135 (46%). Median locAL-PFS after first treatment was 56 months. Responders had longer locAL-PFS (78 vs 17 months; P < .001). Three patients with lung locAL and a MC were diagnosed as systemic AL amyloidosis at follow-up. In summary, locAL pathogenesis seems to be heterogeneous and the clonal infiltrate leads local progression.

Novel challenges in the management of immunoglobulin light chain amyloidosis: from the bench to the bedside.

INTRODUCTION: Immunoglobulin light chain (AL) amyloidosis is one of the most frequent systemic amyloidosis in Western countries. It is caused by a B-cell clone producing a misfolded light chain (LC) that deposits in organs. AREAS COVERED: The review examines recent findings on pathophysiology and clinical management of AL amyloidosis. It contains an update on the recent hot topics as novel therapeutic approaches, definition of relapse, and hematologic response assessment. To review literature on AL amyloidosis, a bibliographic search was performed using PubMed. EXPERT OPINION: Due to the proteotoxicity of amyloidogenic LCs, the therapeutic goal is a rapid and profound decrease in their concentration. The standard treatment is a risk-adapted chemotherapy targeting the B-cell clone. Novel, promising drugs, as daratumumab, are currently under evaluation in newly-diagnosed and relapsed/refractory patients. New sensitive techniques, as mass spectrometry approach and bone marrow minimal residual disease assessment, are available to evaluate depth of response. After first-line therapy, increase in LC concentration may precede worsening of organ dysfunction and should be considered carefully. Further clarification of molecular mechanisms of the disease are shedding light on new possible therapeutic targets. Innovative treatment strategies and novel technologies will improve our ability to treat AL amyloidosis, preventing organ deterioration.

CT features in amyloidosis of the respiratory system - Comprehensive analysis in a tertiary referral center cohort.

PURPOSE: Amyloidosis of the respiratory system is rare and challenging since imaging findings have several more prevalent alternative diagnoses. We analyze and quantify chest CT findings in a large tertiary referral center patient cohort with confirmed amyloidosis of the respiratory system. METHODS: 67 patients with histology-proven amyloidosis of the respiratory system and with available chest CT scans were retrospectively enrolled (years 2002-2018): 41 patients with local pulmonary parenchymal, 20 with local tracheobronchial, and 6 with systemic amyloidosis. CT was scored for findings like mass lesions, nodules, cysts, lymphadenopathy, calcifications and pleural, interstitial and tracheobronchial manifestations. Clinical data and imaging findings' frequencies among patients with local pulmonary parenchymal and tracheobronchial amyloidosis were compared. RESULTS: Patients with local pulmonary parenchymal amyloidosis were older (67 vs. 56 years; P = 0.013) and less frequently symptomatic for cough (24% vs. 70%; P = 0.018) and bronchopulmonal infections (7% vs. 55%; P < 0.001) than patients with tracheobronchial amyloidosis. Local pulmonary parenchymal amyloidosis showed higher frequency of mass-like lesions (41% vs. 0%; P = 0.002) and nodules (95% vs. 20%; P < 0.001, with 10 or more nodules in 56% vs. 0%; P < 0.001 and predominantly pleura-associated in 32% vs. 0%; P = 0.02). Tracheobronchial amyloidosis leads to wall thickening of the bronchi (100% vs. 5%; P < 0.001) and the trachea (70% vs. 2%; P < 0.001). Systemic amyloidosis went along with a predominant alveolar septal pattern in 4 out of 6 patients. CONCLUSION: Patients with local pulmonary parenchymal amyloidosis differ significantly from patients with tracheobronchial amyloidosis regarding clinical data and CT findings' frequencies. Being familiar with radiological manifestations of all three respiratory amyloidosis distribution patterns is essential to accelerate the diagnosis.

Pomalidomide and dexamethasone grant rapid haematologic responses in patients with relapsed and refractory AL amyloidosis: a European retrospective series of 153 patients.

Pomalidomide demonstrated activity in the treatment of AL amyloidosis in three phase II clinical trials. We evaluated the safety and efficacy of 28-day cycles of pomalidomide and dexamethasone in 153 previously treated patients with systemic AL amyloidosis. Ninety-nine (65%) were refractory to the last line of therapy and 54 (35%) had relapsed. The median number of previous lines of therapy was 3 (range: 2-7): 143 patients (93%) previously received bortezomib, 124 (81%) lenalidomide, 114 (75%) oral melphalan, and 37 (24%) underwent autologous stem cell transplant. At the completion of cycle 6, 68 (44%) patients obtained at least partial haematologic response, with 5 complete responses (CR, 3%), 35 very good partial responses (VGPR, 23%). Haematologic response resulted in improved overall survival (median survival 50 vs. 27 months, p = .033) in a 6 months landmark analysis. Obtaining at least partial response was also associated with a significant improvement of the progression-free survival (median PFS 37 vs. 18 months, p < .001). Pomalidomide is an effective treatment for heavily pre-treated patients with AL amyloidosis. Haematologic responses are associated with an overall survival advantage.