OBJECTIVE:To map the occurrence of amyloid types in a large clinical cohort using mass spectrometry-based shotgun proteomics, an unbiased method that unambiguously identifies all amyloid types in a single assay.METHODS:A mass spectrometry-based shotgun proteomics assay was implemented in a central reference laboratory. We documented our experience of typing 16,175 amyloidosis specimens over an 11-year period from January 1, 2008, to December 31, 2018.RESULTS:We identified 21 established amyloid types, including AL (n=9542; 59.0%), ATTR (n=4600; 28.4%), ALECT2 (n=511; 3.2%), AA (n=463; 2.9%), AH (n=367; 2.3%), AIns (n=182; 1.2%), KRT5-14 (n=94; <1%), AFib (n=71; <1%), AApoAIV (n=57; <1%), AApoA1 (n=56; <1%), AANF (n=47; <1%), Aβ2M (n=38; <1%), ASem1 (n=34; <1%), AGel (n=29; <1%), TGFB1 (n=29; <1%), ALys (n=15; <1%), AIAPP (n=13; <1%), AApoCII (n=11; <1%), APro (n=8; <1%), AEnf (n=6; <1%), and ACal (n=2; <1%). We developed the first comprehensive organ-by-type map showing the relative frequency of 21 amyloid types in 31 different organs, and the first type-by-organ map showing organ tropism of 18 rare types. Using a modified bioinformatics pipeline, we detected amino acid substitutions in cases of hereditary amyloidosis with 100% specificity.CONCLUSION:Amyloid typing by proteomics, which effectively recognizes all amyloid types in a single assay, optimally supports the diagnosis and treatment of amyloidosis patients in routine clinical practice.

Gardner syndrome describes a variant phenotype of familial adenomatous polyposis (FAP), primarily characterized by extracolonic lesions including osteomas, dental abnormalities, epidermal cysts, and soft tissue tumors. We describe a 2-yr-old boy presenting with a 2-cm soft tissue mass of the forehead. Pathologic evaluation revealed a nuchal-type/Gardner-associated fibroma. Sequencing of the APC gene revealed a pathologic variant c.4666dupA. Parental sequencing of both blood and buccal tissue supported the de novo occurrence of this pathologic variant. Further imaging revealed a number of additional lesions including a large lumbar paraspinal desmoid, a 1-cm palpable lesion posterior to the left knee, firm lesions on bilateral heels, and multiple subdermal lesions. Colonoscopy was negative. This case illustrates a genetic variant of Gardner syndrome resulting in an aggressive early childhood phenotype and highlights the need for an individualized approach to treatment.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

Purpose CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD. Methods Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects. Results The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression. Conclusions Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.

1501 Background: Despite BC risk reduction of 50-65% by preventive endocrine therapy (ET), very few at-risk women choose to take them. A woman’s perceived BC risk correlates with uptake of ET. A PRS comprised of 77 BC genetic susceptibility loci (Single Nucleotide Polymorphisms (SNP) improves the accuracy of risk prediction for BC. We examined the impact of the addition of individualized PRS BC risk prediction to standard risk calculator estimates on intent to take BC prevention medication. Methods: Eligible women had ≥5% 10 yr BC Tyrer-Cuzick risk (IBIS) or 5 year Gail score ≥3%, with no history of BC or hereditary BC syndrome. Standard BC risk estimates (IBIS or Gail) were incorporated into the counselling on BC preventive ET. A self-reported questionnaire at baseline quantified intention to take ET and explored factors associated with this decision. Blood samples were obtained and genotyped for 77 SNPs, individualized PRS were calculated then incorporated into IBIS and Gail predictions for 5 yr, 10 yr, & lifetime BC risk. At a second visit, PRS risk & prevention recommendations were revisited. Post visit questionnaires assessed change in intent to take ET. Multivariable linear regression was performed to assess impact of baseline variables on change in intent to take medication. Results: From 2016 to 2017, 151 women in Canada & USA were enrolled, median age: 56.1 (range 36-76.4), 35.6% were premenopausal, 98.7% were Caucasian. Median 5yr, 10yr, & lifetime IBIS risk estimates were 3.8% (2.0-11.5), 7.9% (5.0-23.1), and 25.3% (5.5 to 92.2). PRS increased BC risk estimates in 84 (55.6%) and reduced BC risk estimates in 67 (44%) women. After PRS risk counselling, intention to take ET significantly changed (p<0.001): 41.9% of those with increased PRS were more inclined, and 46.7% of women with decreased PRS were less inclined to take ET. On multivariable regression, increase in PRS (p<0.0001) and less concern about ET side effects (p<0.0001) were associated with greater intent to take ET. Conclusions: In high risk women, PRS significantly changed BC risk estimates & intent to take preventive ET. Further assessments of the impact of PRS scores on compliance with ET are warranted. Clinical trial information: NCT02517593.

Pathogenic variants inCOCH, encoding cochlin, cause DFNA9 deafness disorder with characteristic histopathologic findings of cochlin deposits in the inner and middle ears. Here, we present the first case of deafness associated with bilateral external auditory canal (EAC) cochlin deposits, previously unreported evidence suggestive of cochlin-derived amyloid formation, and a novelCOCHvariant. A 54-year-old woman presented with progressive sensorineural hearing loss and bilateral EAC narrowing by subcutaneous thickening. Excision and histologic evaluation of tissue from both EACs showed paucicellular eosinophilic aggregates containing multiple Congo red-positive foci with yellow and green birefringence under crossed polarization light microscopy. Mass spectrometry performed on both the Congo red-positive and Congo red-negative areas identified cochlin as the most abundant protein, as well as a low abundance of universal amyloid signature peptides only in the Congo red-positive areas. Peptides indicative of a canonical amyloid type were not detected. Electron microscopy showed haphazard, branched microfibrils (3-7 nm in diameter) consistent with cochlin, as well as swirling fibrils (10-24 nm in diameter) reminiscent of amyloid fibrils. Cochlin immunohistochemical staining showed positivity throughout the aggregates. Sequencing of the entireCOCHgene coding region from the patient's blood revealed a novel variant resulting in a non-conservative amino acid substitution of isoleucine to phenylalanine (c.1621A>T, p.I541F) in the vWFA2 domain at the protein's C-terminus. Our findings reveal a new pathologic manifestation of cochlin, raise the possibility of previously undescribed cochlin-derived amyloid formation, and highlight the importance of thoroughly investigating all aggregative tissue findings in the practice of diagnostic pathology.

BACKGROUNDDroplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need.METHODSWe validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies.RESULTSFor the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals.CONCLUSIONSStandard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

The recent advances in next-generation sequencing (NGS) technologies have enabled the development of effective high-throughput noninvasive prenatal screening (NIPS) assays for fetal genetic abnormalities using maternal circulating cell-free DNA (ccfDNA). An important NIPS quality assurance is quantifying the fetal proportion of the sampled ccfDNA. For methods using allelic read count ratios from targeted sequencing of single-nucleotide polymorphisms (SNPs), systematic biases and errors may reduce accuracy and diminish assay performance. We collected ccfDNA NIPS MiSeq sequencing data from an amplicon-based 92 SNP panel along with complementary low-depth whole-genome sequencing (WGS) on 243 normal male fetus pregnancies along with additional 144 nonpregnant female donor samples. Using fetal fraction estimates based on X and Y chromosome WGS coverage as gold standard, we compared an existing SNP-based approach, FetalQuant, to a more flexible Bayesian hierarchical modeling strategy that borrows information across interrogated SNPs to character SNP-level error rates and biases to improve fetal fraction estimates. Posterior distributions for SNP-level model parameters indicate most SNPs exhibited modest to moderate extrabinomial variation and a consistent underrepresentation of fetal alleles, with some extreme outliers in both regards. Fetal fraction estimates using FetalQuant, naive to these SNP properties, were relatively poor (R-2 = 0.14, root mean squared error [RMSE] = 0.050), particularly when the true fetal fraction was low (<5%). In contrast, by quantifying SNP-level biases and error rates, our proposed approach demonstrated improved performance by reducing the bias and variability in fetal fraction estimates (R-2 = 0.794, RMSE = 0.025). Using high-depth targeted SNP sequencing data, we identified a high degree of variability in distributional properties across SNP allelic read counts. These results highlight the benefits of leveraging hierarchical modeling for SNP-based fetal quantification assays (FQAs) and the need to properly calibrate FQAs dependent on NGS allelic ratio data.

Hereditary amyloidosis represents a group of diseases in which mutant proteins are deposited in various organs leading to their dysfunction. Correct identification of the amyloid-causing protein is critical because this will determine the optimal therapy for the patient. The most common type of hereditary amyloidosis is due to mutant transthyretin (ATTRm) deposition and often presents with heart failure or peripheral neuropathy. We report the first known case of a patient who had amyloidosis both due to a mutant transthyretin (p.Val122Ile) and due to a novel variant in the gelsolin gene (p.Ala578Pro). Both mutant proteins were identified by mass spectrometry analysis of amyloid deposits as well as sequencing of the genes. Molecular dynamic simulations suggest that the gelsolin p.Ala578Pro variant is likely amyloidogenic.

PURPOSE:To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD). METHODS:Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients. RESULTS:The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10). No significant association was detected between the TNR length and clinical parameters. CONCLUSIONS:Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.

Fibrillary GN (FGN) is a rare primary glomerular disease. Histologic and histochemical features of FGN overlap with those of other glomerular diseases, and no unique histologic biomarkers for diagnosing FGN have been identified. We analyzed the proteomic content of glomeruli in patient biopsy specimens and detected DnaJ heat shock protein family (Hsp40) member B9 (DNAJB9) as the fourth most abundant protein in FGN glomeruli. Compared with amyloidosis glomeruli, FGN glomeruli exhibited a >6-fold overexpression of DNAJB9 protein. Sanger sequencing and protein sequence coverage maps showed that the DNAJB9 protein deposited in FGN glomeruli did not have any major sequence or structural alterations. Notably, we detected DNAJB9 in all patients with FGN but not in healthy glomeruli or in 19 types of non-FGN glomerular diseases. We also observed the codeposition of DNAJB9 and Ig-γ Overall, these findings indicate that DNAJB9 is an FGN marker with 100% sensitivity and 100% specificity. The magnitude and specificity of DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN pathogenesis. With this evidence, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsy specimens.

BACKGROUND & AIMS: Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. METHODS: We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. RESULTS: Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a >= 2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 +/- 0.82 patients with tumoremia vs 0.64 +/- 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. CONCLUSION: In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

BACKGROUND Most patients who undergo resection for pancreatic ductal adenocarcinoma (PDAC) ultimately develop local recurrence and/or distant metastasis that are largely attributed to occult disease not detected during pre-operative staging or surgery.These cells and nuclear material can be detected in the blood as circulating tumor cells (CTCs) or cell-free DNA (cfDNA) as a means of "liquid biopsy" and their presence is referred to as tumor seeding (tumoremia).While occult dissemination of tumor cellular and nuclear material into the bloodstream is well-recognized, the risk of procedure-induced tumor seeding has not been established.In PDAC patients, we aimed to prospectively evaluate peripheral, portal vein (PV), and hepatic vein (HV) blood to determine the risk of endoscopic ultrasound guided fine needle aspiration (EUS FNA)-induced tumoremia.METHODS Peripheral blood from 104 patients (35 PDAC, 69 healthy controls) and PV and HV blood from 10 PDAC patients were obtained before and following primary tumor FNA.Plasma cell free deoxyribonucleic acid (cfDNA) concentrations were measured and V-Ki-ras2 Kirsten rat sarcoma (KRAS) 12/13 mutation status was evaluated in cfDNA and primary tumors.Tumoremia was defined as a $2-fold increase in cfDNA and/or conversion from circulating tumor deoxyribonucleic acid (ctDNA) KRAS mutant non-detectable to detectable.RESULTS Based on pre-defined metrics using digital droplet (dd) PCR [ $2-fold increase in cfDNA concentration (n=7) and/or conversion from ctDNA-wild type to mutant status (n=3)], FNA-induced tumoremia occurred in 10 (28.6%) patients.The peripheral blood cfDNA concentration pre-EUS FNA was significantly higher for the PDAC patients than controls [1,200 ng/ml (500-3,300) vs. 600 ng/ml (475-825); p=0.0013].The median PV plasma cfDNA concentration displayed a non-significant increase following primary tumor FNA [1,100 ng/ml (430-3,210) versus 1,300 ng/ml (320-3,010); p=0.853].No patient developed evidence of hemorrhage.CONCLUSIONS Our data suggest that primary tumor FNA releases nuclear material into the circulation and potentially induces tumoremia.We demonstrated the feasibility and safety of serial PV and HV blood collections.Studies are needed to confirm our results and to assess the impact upon patient outcome, to establish the benefit of quantitative and qualitative cfDNA/ctDNA testing, and to clarify the utility of sampling central blood.PDAC patients lack personalized treatment options due to the absence of effective targeted therapies and because tumor biopsy provides incomplete molecular characterization.While liquid biopsy may overcome these limitations, our data suggest that currently cfDNA analysis cannot replace tumor biopsy for diagnosis or consistently detect actionable mutations for individualized patient care.

Occult dissemination of tumor cellular and nuclear material into the bloodstream is well-recognized and has been reported for a variety of tumor types, including pancreatic ductal adenocarcinoma (PDAC). These cells and nuclear material can be detected in the blood as circulating tumor cells (CTCs) or cell-free DNA (cfDNA) as a means of "liquid biopsy" and their presence is referred to as "tumoremia." Limited data suggest that various biopsy procedures and surgery can also precipitate tumoremia. There are no published data indicating the risk of hematogenous tumor seeding following EUS FNA and no data clarifying whether any factors may be associated with its occurrence. In PDAC patients, we aimed to prospectively evaluate peripheral blood to determine the risk of EUS FNA-induced tumoremia and to identify the associated clinical, laboratory, and tumor demographic features. Peripheral blood from 35 patients with PDAC was collected before and following primary tumor EUS FNA. Plasma cfDNA concentrations and KRAS 12/13 mutation status were evaluated. Tumor seeding was defined by a ≥2-fold increase in cfDNA and/or conversion from circulating tumor deoxyribonucleic acid (ctDNA) KRAS mutant non-detectable to detectable. Factors that were evaluated to determine their correlation with tumoremia included age, gender, BMI, ECOG status, serum glucose level, CA 19-9 concentration, tumor location, tumor diameter, tumor stage, resectability status, number of FNA passes, infiltration of major arteries and veins, presence of IPMN, mortality, survival duration, pre-FNA cfDNA level, and the tumor allelic ratio. Based on our pre-defined metrics, either a ≥2-fold increase in the total cfDNA concentration (n=7; 20.6%), and/or conversion in ctDNA mutant detection status (n=3; 8.8%) primary tumor EUS FNA-induced hematogenous tumor seeding occurred in 10/35 (28.6%) patients. A broad spectrum of clinical, laboratory, and tumor demographic features were evaluated, none of which statistically correlated with a ≥2-fold increase in cfDNA, other than the number of major arteries and/or veins infiltrated by the primary tumor. (Table) The only feature that correlated with the conversion in KRAS mutation status was the CA 19-9 level. For the combined group (n=10), who met either metric for FNA-induced tumoremia, there were no characteristics that correlated with these endpoints. Our data indicate that the occurrence of hematogenous tumor seeding associated with EUS FNA at the present time, cannot be predicted based upon an evaluation of an extensive array of clinical, laboratory, and tumor related characteristics. Additional studies are needed to determine the clinical impact of EUS FNA-induced tumor seeding as well as to verify whether any predictive features can be identified to help predict its occurrence.Tabled 1≥2 fold increase in cfDNA concentrations &/or ctDNA- WT to ctDNA-MT Status (n=10)Remaining Cohort (n=25)P ValueAge (years)70.6 ± 6.269.4 ± 9.20.699Gender (female)4 (40.0%)14 (56.0%)0.471BMI24.8 ± 5.227.8 ± 6.90.229ECOG (≥1)1 (10.0%)9 (36.0%)0.407Glucose level (median; mg/dL)105.0 (98.0-107.0)121.5 (104.5-146.5)0.089Ca19-9 (median; U/mL)374.0 (222.0-1701.0)581.0 (74.5-1617.5)0.969Ca19-9 (>55 U/mL)9 (90.0%)19 (76.0%)0.645Tumor location (head)8 (80.0%)20 (80.0%)0.9Tumor diameter (long axis; mm)32.5 ± 6.531.9 ± 9.70.863Tumor diameter (short axis; mm)28.4 ± 7.026.4 ± 7.80.481Tumor stage (III/IV)5 (50.0%)11 (44.0%)0.9Tumor stage (I/II/III)8 (80.0%)18 (72.0%)0.9Tumor (resectable/borderline)5 (50.0%)14 (56.0%)0.9EUS FNA passes (median)5.5 (4.0-8.0)5.0 (4.8-6.0)0.739Infiltrate major artery (yes)4 (40.0%)11 (44.0%)0.9Infiltrate major vein (yes)4 (40.0%)15 (60.0%)0.454Infiltrate major artery or vein (yes)6 (60.0%)16 (64.0%)0.9# of major arteries/veins involved1.0 (0.0-2.0)2.0 (0.0-3.0)0.321Adenocarcinoma (non-IPMN)7 (70.0%)18 (72.0%)0.9Mortality (yes)6 (60.0%)14 (56.0%)0.9Time to mortality (median; months)6.4 (4-12.3)7.2 (4.4-11.3)0.9Perip Pre-FNA cfDNA level (ng/ml)0.6 (0.2-2.8)1.3 (0.6-3.5)0.201Allelic ratio (median; primary tumor)10.4 (1.5-23.4)18.9 (5.3-28.9)0.317 Open table in a new tab

Context.-The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing. Objective.-To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3. Design.-A proficiency testing sample with discordant results for spinocerebellar ataxia type 3 analysis was further evaluated by targeted Sanger sequencing and genotype polymerase chain reaction using multiple DNA polymerases. Results.-Of 28 laboratories responding in the spinocerebellar ataxia type 3 Proficiency Survey, 18 reported an incorrect homozygous result and 10 reported the expected heterozygous result. A heterozygous single-nucleotide polymorphism complementary to the 3' end of a published forward primer was identified in the proficiency testing sample, which may have led to allele dropout. However, this primer was used by only 3 of 18 laboratories (16%) reporting a homozygous result. A new forward primer of identical sequence, except for the 3' end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3'-5' exonuclease activity contributing to allele dropout was investigated by testing 9 additional polymerases with and without exonuclease activity. No clear pattern emerged, but enzymes with and without 3'-5' exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer. Conclusions.-Proactive systematic primer sequence checking is recommended because single-nucleotide polymorphism interference may result in allele dropout and impact clinical testing. Allele dropout is also influenced by other factors, including DNA polymerase exonuclease activity.

Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited nodular glomerular involvement. Although prior in vitro studies have shown that Apo-CII can form amyloid fibrils and that certain mutations in this protein promote amyloid fibrillogenesis, there are no reports of this type of amyloidosis in humans. We propose that this study reveals a new form of hereditary amyloidosis (AApoCII) that is derived from the Apo-CII protein and appears to manifest in the elderly and preferentially affect the kidneys.