BACKGROUND:Cardiac amyloidosis, primarily due to immunoglobulin light chain (AL) or transthyretin (ATTR) amyloid, is an increasingly recognized cause of heart failure. Modern diagnostic advances suggest that ATTR, particularly in older adults, may be more prevalent than historically reported. METHODS:All Olmsted County decedents aged ≥40 years from 1970 to 1976 were identified. Available ventricular myocardium from retained paraffin blocks was screened histologically for amyloid using sulfated Alcian blue staining; positive cases underwent grading and proteomic typing by laser microdissection coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Beyond prevalence estimation, this analysis characterizes amyloid type, deposition grade and distribution, associated comorbidities, and cause-of-death patterns, comparing amyloid-positive decedents with age- and sex-matched controls. RESULTS:Of 2,566 eligible deaths, 1,028 autopsy cases with evaluable myocardium formed the study cohort (mean age 70.5 years; 61.1% male; 97% White). Cardiac amyloid was present in 52 cases giving an overall prevalence of 5.1% (95% CI: 3.8-6.6, which rose from 0% under age 60 to 37.5% (95% CI: 21.1-56.3) in those ≥90 years (p < 0.001). While prevalence estimates were higher in men above age 80 compared to women, there was no evidence of an interaction of age and sex (p = 0.90). The quantity of amyloid was sufficient for typing in 38 cases: ATTR (84.2%), AL (7.9%), serum amyloid A (5.3%), and apolipoprotein A-IV (2.6%). Adjusted estimates assuming untyped mild cases were ATTR-type increased ATTR prevalence to 4.5% (95% CI: 3.3-5.9) overall. Comorbidity profiles were similar between amyloid-positive and negative groups, though syncope and leg weakness were more common in amyloid-positive decedents. CONCLUSIONS:In an unselected autopsy cohort, cardiac amyloid was common, particularly ATTR in older adults. Prevalence increased steeply after age 80. These findings suggest that ATTR amyloidosis is not rare and is likely underdiagnosed and has similar prevalence in women and men, despite the male predominance reported in the literature.

Although next-generation sequencing (NGS) has become the standard of care in myeloid neoplasms, the utility of lymphoma NGS (LNGS) in the clinical setting has not been well established. The goal of this study was to document the real-world use of LNGS panels and assess their prognostic, predictive, and diagnostic value. A total of 384 cases were retrospectively reviewed in conjunction with pathology reports, including 121, 183, and 80 tested for the limited B-cell lymphoma panel (25 genes), expanded B-cell lymphoma panel (46 genes), or T-cell lymphoma panel (22 genes), respectively. Specimen types included 170 (44.3%) bone marrow, 120 (31.3%) peripheral blood, and 93 (24.2%) formalin-fixed paraffin-embedded tissue. 248/384 (64.6%) cases showed at least one mutation; 245/347 (70.6%) when excluding specimens known to be negative for lymphoma. Chronic lymphocytic leukemia/small lymphocytic lymphoma (SLL/CLL, 40.6%) and mantle cell lymphoma (MCL, 9.6%) were the most common lymphoma types tested. Mutations with prognostic value were found in 228/384 (59.4%) cases. 125 (32.6%) tests provided predictive value for response or resistance to a specific therapy. 36 tests (9.4%) provided diagnostic value, aiding the pathologist in classifying disease or rendering a more definitive diagnosis. LNGS results are most useful as being prognostic in CLL/SLL and MCL, diagnostic in angioimmunoblastic T-cell lymphoma and hairy cell leukemia, theranostic in lymphoplasmacytic lymphoma, and diagnostic and prognostic in follicular lymphoma and T/NK-cell large granular lymphocytic leukemia. Although clinical guidelines have not yet widely incorporated LNGS, when carefully designed and selectively ordered by clinicians and pathologists, the majority of tests do offer prognostic and predictive data which can aid in optimization of clinical management. The diagnostic value of LNGS testing is likely to increase with its further use and availability.

Apolipoprotein A-I (apoA-I), the major structural and functional protein of plasma high-density lipoprotein (HDL), mediates cholesterol removal from the body and has other beneficial properties. APOA1 gene mutations can cause diseases including human hereditary AApoAI amyloidosis, a life-threatening multiorgan disorder caused by apoA-I misfolding and amyloid deposition. We report esophageal AApoAI amyloidosis in a 66-year-old patient. Amyloid fibrils are comprised of a novel p.Trp96Arg (W72R) apoA-I variant identified by protein mass spectrometry and gene sequencing. Both variant and wild-type apoA-I circulate in plasma at low HDL levels typical of this dominant negative disorder. To understand why W72R substitution promotes amyloid formation, we generated recombinant full-length W72R and wild-type proteins and compared their structural, stability and ligand-binding properties using spectroscopic (circular dichroism, fluorescence, absorption) and biochemical approaches. High-resolution structures of apoA-I in native and in amyloid states were harnessed to propose structural underpinning for the pro-amyloidogenic effects of W72R substitution. W72R substitution slightly destabilized native lipid-free protein; decreased structural protection of amyloid-promoting regions; accelerated lipid solubilization by apoA-I and destabilized model lipoproteins; facilitated apoA-I binding to heparin (a proxy for heparan sulfate) and to type-I collagen; accelerated amyloid formation and potentially stabilized the amyloid structure. All these effects are pro-amyloidogenic. The results help explain the pro-amyloidogenic nature of this mutation, suggest apoA-I binding to extracellular matrix components such as heparan sulfate and collagen as therapeutic targets, and expand our knowledge of the clinical presentation and molecular basis of this complex disease.

BACKGROUND:Advances in fibril typing by mass spectrometry have improved the accuracy of amyloidosis diagnosis. Dual amyloidogenic proteins have been reported in deposits in sole and multiple different organs. METHODS:Five patients with dual amyloidoses were diagnosed between 1995 and 2022 by Congo red staining and fibril typing using the best available methods at the time of evaluation. Sequencing of TTR and GSN genes was performed. Literature search identified 46 additional cases. RESULTS:Three patients exhibited Waldenström macroglobulinemia-associated AL (n = 3) amyloidoses in conjunction with ATTRwt or AGel amyloidosis; two patients featured AL/ATTRwt and AA/ATTRwt amyloidoses. One patient demonstrated dual amyloidoses within one anatomical site; three patients featured two amyloidosis types at different anatomical sites; and one patient had dual amyloid deposits in a single anatomical site along with different sites. The time interval between diagnoses was 0-288 months, with the heart and kidneys being the most affected organs. CONCLUSIONS:Our findings underscore the complexity of clinical presentation in amyloidosis, as multiple amyloid types can co-exist in a single individual and affect various anatomical sites. Accurate assessment of the clinical phenotype and thorough amyloid fibril typing from the target organs are essential for precise diagnosis and tailored treatment. TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT00898235.

Context.—:Treatments are available for common forms of systemic amyloidosis that show promise for extending and improving the quality of life for patients. Early diagnosis and accurate identification of amyloid fibril type are crucial for successful treatment, but the diagnosis and workup of amyloidosis is inconsistent among pathologists and laboratories. Thus, the goal of this guideline is to offer recommendations for proper testing and workup for amyloidosis to optimize patient care. Objective.—:To establish evidence-based recommendations for appropriate laboratory testing to detect amyloidosis and identify the specific amyloidogenic protein. Design.—:The College of American Pathologists convened a panel of experts to develop recommendations following the standards established by the National Academy of Medicine for developing trustworthy clinical practice guidelines. The panel conducted a systematic literature review addressing 6 key questions. Using the Grading of Recommendations Assessment, Development and Evaluation framework, recommendations were created based on the available evidence, certainty of that evidence, and key judgments as defined in the framework. Results.—:Four conditional recommendations and 3 good practice statements were established to provide guidance for proper testing and workup of amyloidosis. Conclusions.—:This guideline summarizes the available evidence on the diagnosis and workup of systemic amyloidosis in tissue samples, including the challenges and limitations of common approaches and techniques. Recommendations for pathologists and laboratories receiving these samples are provided.

Intensified chemoimmunotherapy regimens are often used in young patients with double-hit and triple-hit lymphoma (DHL/ THL) despite no survival benefit compared to R-CHOP. Favorable retrospective reports on the application of CODOX-M/IVAC-R are subject to selection bias as only young fit patients can tolerate this treatment. We conducted a retrospective analysis to investigate outcome differences between CODOX-M/IVAC-R and DA-EPOCH-R in DHL/THL patients aged 60 years or younger. One hundred and thirteen patients were identified; CODOX-M/IVAC-R (N=49) and DA-EPOCH-R (N=64). Eighty percent (39/49) achieved complete (CR) after completing CODOX-M/IVAC-R compared to 58% (37/64) with DA-EPOCH-R. The median follow-up was 5.3 years and 3.3 years for the CODOX-M/IVAC-R and DA-EPOCH-R group respectively. CODOX-M/IVAC-R demonstrated superior event-free survival (EFS) on univariate (hazard ratio [HR]=0.54, 95% confidence interval [CI]: 0.31-0.97) and multivariable analysis adjusted for age, BCL translocation (BCL2 vs. BCL6 vs. both), International Prognostic Index score and receipt of autologous stem cell transplant (adjusted HR [aHR]=0.52, 95% CI: 0.29-0.93); however there was no significant influence on OS (aHR=0.92, 95% CI: 0.46-1.84). The 1, 2 and 5 years EFS in the CODOX-M/IVAC-R group was 68.3%, 64.1% and 61.5%, respectively compared to 52.4%, 48.9% and 39.5%, respectively in the DA-EPOCH-R group. Primary refractory disease or relapse (R/R) occurred in 33% (16/49) of CODOX-M/IVAC-R and 54% (35/64) of DA-EPOCH-R recipients, and produced median OS of 10.3 months and 33.7 months, respectively, indicating poor outcomes in the CODOX-M/IVAC-R subgroup with R/R disease. More patients were able to receive subsequent salvage therapies in the DA-EPOCH-R group. No patients died of regimen toxicity and the rates of central nervous system relapse and therapy related hematologic neoplasms were similar in both groups.

BACKGROUND:Cardiac AL and ATTR are potentially fatal cardiomyopathies. Current therapies do not address mechanisms of tissue dysfunction because these remain unknown. Our prior work focused on the amyloid plaque proteome, which may not capture tissue-wide proteomic alterations. OBJECTIVES:To evaluate mechanisms of tissue dysfunction in cardiac AL and ATTR using a full biopsy tissue proteomics approach. METHODS:We performed proteomics analysis on 76 ATTR and 27 AL diagnostic endomyocardial biopsies. RESULTS:Stage-3 AL patients exhibited increased coagulation, extracellular matrix remodelling (ECM), epithelial-to-mesenchymal transition (EMT), complement activation, hypoxia, and clathrin-mediated endocytosis pathways vs. stages-1/2, with decreased healthy cardiac metabolism. In stages-2 and 3 ATTR, immunoglobulin proteins, complement, and keratinisation pathways were increased compared to stage-1. Unsupervised analyses identified an ATTR group with worse survival characterised by upregulated complement and downregulated metabolic pathways. Compared to ATTR, AL had higher clathrin-mediated endocytosis, mRNA splicing, and ribosomal proteins, while ATTR had higher complement levels. CONCLUSIONS:This study identifies known processes dysregulated in heart failure with preserved ejection fraction as well as novel pathways responsible for tissue damage. Our results support an immune-mediated mechanism of tissue toxicity in cardiac amyloidosis, especially among patients with worse outcomes.

Wild-type transthyretin (ATTRwt) amyloidosis typically presents with restrictive cardiomyopathy. Kidney involvement is exceedingly rare. We report to our knowledge the first antemortem diagnosis of ATTRwt amyloidosis with kidney vasculature deposition in a patient presenting with progressive kidney failure. Notably, technetium-99m pyrophosphate scintigraphy showed no myocardial uptake. This case expands the known spectrum of organ involvement in ATTRwt amyloidosis and underscores the need to consider extracardiac manifestations in its diagnosis.

BACKGROUND:Patients with two different amyloid types are rare. It is critical to identify all types of amyloid in a patient as amyloid therapies vary dramatically depending on the specific precursor protein. OBJECTIVE:To analyze the clinicopathologic and proteomic features in patients with two types of amyloid. METHODS:We queried our reference laboratory database of 51,309 amyloid specimens from all anatomic sites typed by mass spectrometry-based proteomics (LC-MS/MS) for patients diagnosed with two different amyloid types. Demographic data (patient age, sex, and anatomic site), mass spectrometry proteomic features, and clinical history, when available, were reviewed. RESULTS:We identified 111 patients with two amyloid types: 83 male (75 %), 28 female (25 %). The median age at initial diagnosis was 78 years (range 45-96 years). 56 patients (50 %) had cardiac involvement. The two amyloid types were present either in the same anatomic compartment within a single specimen (48 patients, 43 %), in different anatomic compartments within a single specimen (43 patients, 39 %), or in two different anatomic locations (20 patients, 18 %). The most common combination was AL plus ATTR (69 patients, 62 %), which occurred in 68 % of patients with cardiac involvement. CONCLUSION:It is crucial to identify all amyloid types in all cases, including careful morphologic review to evaluate for distinct areas with different distribution, and consider the possibility of a second amyloid type when the clinical findings are not explained by the initial amyloid type. In some patients, this may require biopsy from a second anatomic site.

Apolipoprotein A-I (apoA-I) plays beneficial roles as the major structural and functional protein on plasma high-density lipoproteins (HDL). However, APOA1 gene mutations can cause protein misfolding and pathologic amyloid deposition in various organs in human hereditary AApoAI amyloidosis, a potentially lethal systemic disease. We report esophageal and duodenal AApoAI amyloidosis in a 56-year-old patient with Barrett's esophagus, a condition involving chronic acid reflux. Amyloid deposits contained full-length apoA-I featuring a novel D20Y mutation identified by gene sequencing and protein mass spectrometry. Genetic analysis of asymptomatic family members revealed autosomal dominant inheritance. Fibril formation by the full-length variant apoA-I rather than its fragments and the location of the mutation in a conserved amyloid-prone N-terminal segment were highly unusual for hereditary AApoA-I amyloidosis. Structural and stability studies of the recombinant D20Y and wild-type apoA-I showed small but significant mutation-induced structural perturbations in the native lipid-free protein at pH 7.4. Major destabilization and aggregation of the variant protein were observed at pH 4.0. We propose that acidic conditions in Barrett's esophagus promoted protein misfolding and amyloid formation by the D20Y variant. These findings expand our understanding of the clinical features and molecular basis of AApoAI amyloidosis and suggest clinical strategies.

Lymphocytic variant of hypereosinophilic syndrome (LV-HES) is a rare T-cell lymphoproliferative disorder characterized by an immunophenotypically abnormal Th2 T-cell clone which produces eosinophilopoietic cytokines, resulting in eosinophilia and end-organ damage. A 38-year-old woman presented to an outside institution with a 10-year history of a pruritic, recurrent, steroid-responsive skin eruption and a 3-year history of mild lymphadenopathy. Excisional lymph node biopsy demonstrated a clonal, surface CD3-CD4+ T-cell infiltrate, prompting a diagnosis of peripheral T-cell lymphoma, not otherwise specified. Further workup revealed bone marrow and peripheral blood involvement. She received multiagent chemotherapy with temporary resolution of her skin eruption and lymphadenopathy, but persistent bone marrow disease. Presenting to our institution 3 years later, she exhibited numerous flesh-colored papules involving the extremities, without patches or plaques of mycosis fungoides. Skin biopsies demonstrated a dermal perivascular and interstitial proliferation of monotonous small T-cells without significant epidermotropism. T-cell receptor gene rearrangement studies of skin and peripheral blood specimens revealed identical clonal peaks, and peripheral blood flow cytometry showed persistence of the previously identified T-cell clone. Laboratory workup demonstrated a markedly elevated IgE level (66,580 kU/L) with a normal eosinophil count and IL-5 level. Next-generation sequencing of a peripheral blood sample revealed a pathogenic STAT3 S614R variant, previously documented in LV-HES. Although lacking eosinophilia, the patient’s indolent course, characteristic skin lesions, steroid responsiveness, and pathologic features are typical of LV-HES, and the elevated IgE and STAT3 activation underscore a similar biology. We thus propose that this case expands the spectrum of indolent Th2-T cell lymphoproliferative disorders that need to be distinguished from peripheral T-cell lymphoma clinically.

Amyloidosis is a group of disorders characterized by abnormal deposition of amyloid proteins in various tissues and organs, leading to progressive organ dysfunction. With over 40 precursor proteins linked to amyloid formation, identification of the amyloid type is critical to guide treatment. A man in his late 40s presenting with heart failure was diagnosed with cardiac amyloidosis based on an endomyocardial biopsy. Amyloid typing performed on the heart biopsy at Mayo Clinic Laboratories using differential laser microdissection and shotgun proteomics with mass spectrometry reported gelsolin amyloid (AGel) deposits exclusively in the vasculature and transthyretin amyloid deposits exclusively within the interstitium. Mutational analysis identified a novel p.Y474N in the gelsolin gene, establishing a diagnosis of hereditary AGel amyloidosis. Transthyretin gene mutations were absent, confirming a concurrent diagnosis of acquired transthyretin wild-type amyloidosis. The patient, who had been treated with guideline-directed medical therapy since his initial presentation, was subsequently started on tafamidis, with subsequent improvement of his ejection fraction after 6-7 months. Although rare, 2 different amyloid types may arise in the same anatomic site. Identification of all amyloid types is crucial for optimal patient management. In this case, the co-existence of 2 rare amyloid types (AGel with a novel mutation coupled with ATTRwt in a patient under 50 years of age) in mutually exclusive anatomic compartments in the same cardiac biopsy raises the possibility that an unknown systemic factor may play a role in amyloidogenesis in dual amyloid cases.

OBJECTIVE:The aim of this study was to evaluate a large cohort of gallbladder amyloid cases to determine clinical and morphologic features. METHODS:Cholecystectomy specimens (N = 118) typed using proteomics-based techniques between 2008 and 2023 were identified. Clinical and morphologic features were reviewed. RESULTS:Six amyloid types were identified: ATTR (n = 63, 53.4%), AL (n = 46, 39.0%), AA (n = 4, 3.4%), AApoA1 (n = 2, 1.7%), ALECT2 (n = 2, 1.7%), and AEFEMP1 (n = 1, 0.8%). Amyloidogenic mutations were detected in 3 ATTR cases and 2 AApoA1 cases. Morphologic review (n = 26) revealed perimuscular vessel involvement in all cases. Amyloidosis was an unexpected diagnosis first made on the cholecystectomy specimen in half of the patients with clinical information (n = 10). All 9 patients with follow-up had evidence of systemic disease. In 2 patients, cholecystic involvement was initially missed and only retrospectively identified after the diagnosis of cardiac amyloidosis. CONCLUSIONS:In patients with clinical data, amyloidosis was often unexpected, the gallbladder was commonly the first tissue sampled with amyloidosis, and all patients had systemic disease. Thorough review of cholecystectomy specimens with careful inspection of perimuscular vessels, coupled with a low threshold for ordering Congo red stain in elderly individuals and amyloid typing using a robust method such as proteomics, can prevent a delay in amyloid diagnosis and management.