The novel form of amyloidosis derived from EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) preferentially affects the lower gastrointestinal tract of elderly femalesa.

AIMS: To characterise the clinicopathological features of amyloidosis due to EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), a newly described amyloid type. METHODS AND RESULTS: We identified cases by searching the Mayo Clinic amyloid liquid chromatography and tandem mass spectrometry typing database for specimens with the universal amyloid signature proteins, abundant EFEMP1 spectra and absence of other specific amyloid precursor proteins. We also developed an immunohistochemical stain for EFEMP1 applicable to formalin-fixed tissue sections and performed electron microscopy in one case. We identified 33 specimens from 32 patients with EFEMP1 amyloid. Most patients were female (91%) with a mean age of 75 years, and most specimens (94%) were from the bowel. EFEMP1 amyloid was incidentally identified in specimens biopsied/resected for a variety of clinical indications. In bowel specimens, EFEMP1 amyloid involved blood vessels and interstitium of the lamina propria, submucosa and/or muscularis propria. Although the EFEMP1 deposits were weakly to moderately Congo red-positive with absent to weak birefringence, they were strongly positive for EFEMP1 by immunohistochemistry, had the characteristic fibrillar ultrastructure of amyloid and were readily identified by mass spectrometry. CONCLUSIONS: EFEMP1 amyloid is a recently described novel amyloid type that predominantly affects the bowel of elderly females. Because EFEMP1 amyloid is only weakly Congo red-positive, it may be overlooked without a high index of suspicion. However, its characteristic microanatomical distribution is highlighted by immunohistochemistry and its identity is readily confirmed by mass spectrometry. Based on its distinctive features, we propose that EFEMP1 amyloidosis be considered a new amyloid type.

Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease.

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug-protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49-129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25-34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

Amyloidosis in surgically resected atrial appendages: a study of 345 consecutive cases with clinical implications.

Histomorphologic parameters of atrial appendages removed during the Cox-Maze procedure have been shown to correlate with recurrence of atrial fibrillation. While amyloid deposition has been noted within atrial appendages, the incidence and significance remains incompletely understood. More accurate amyloid typing methodologies and targeted pharmacotherapeutics have recently been developed, prompting pathologists to provide more detailed information about the type of amyloid identified in such samples. This study sought to fully characterize the morphologic characteristics of atrial amyloid as well as its incidence and clinical significance. Tissue archives were queried for atrial appendages removed during the cardiac surgeries (2010-2014). Patient demographics, imaging features, and salient clinical findings were recorded. Pattern and extent of amyloid deposition were recorded. Typing of the amyloid protein, when present, was performed on a subset of cases by laser capture microdissection with mass spectrometry-based proteomic analysis. A total of 383 atrial appendages from 345 consecutive patients were included in the study (mean age, 69 years; range, 26-92 years). Amyloid was present in 46% of patients. A linear relationship was observed between age and presence of atrial amyloidosis. Women were more likely to have atrial amyloidosis. Two distinct morphologies of amyloid were observed: filamentous and nonfilamentous, and correlated perfectly with amyloid type (filamentous = AANF-type amyloid; nonfilamentous = ATTR-type amyloid). Filamentous deposits were observed in 91% of those with amyloid. Amyloid was more likely to be found in the left atrial appendage than the right. Patients with atrial amyloid, irrespective of type, were more likely to have experienced stroke or TIA and more likely to have atrial arrhythmia preoperatively. Postoperatively, those with atrial amyloid are more likely to experience recurrence of arrhythmia than those who did not have atrial amyloid. Understanding the morphologic characteristics of AANF-type amyloid will allow for identification by the light microscopy and obviates the need for expensive ancillary typing techniques. The finding of nonfilamentous amyloid, should still prompt confirmation of amyloid type so that targeted therapy may be employed.

Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients.

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease. Kidney biopsy is required to establish the diagnosis. Recent studies have identified abundant glomerular deposition of DNAJB9 as a unique histological marker of FGN. We developed an immunoprecipitation-based multiple reaction monitoring method to measure serum levels of DNAJB9. We detected a 4-fold higher abundance of serum DNAJB9 in FGN patients when compared to controls, including patients with other glomerular diseases. Serum DNAJB9 levels were also negatively associated with estimated glomerular filtration rate in patients with FGN. Serum DNAJB9 levels accurately predicted FGN with moderate sensitivity (67%) and with high specificity (98%) and positive and negative predictive value (89% and 95%, respectively). A receiver operating curve analysis demonstrated an AUC of 0.958. These results suggest that serum levels of DNAJB9 could be a valuable marker to predict FGN, with the potential to complement kidney biopsy for the diagnosis of FGN.

The sensitivity and specificity of the routine kidney biopsy immunofluorescence panel are inferior to diagnosing renal immunoglobulin-derived amyloidosis by mass spectrometry.

Immunoglobulin light chain amyloidosis is the most frequent type of renal amyloidosis in the United States, accounting for 81% of cases. Accurate typing is crucial for early diagnosis and treatment of immunoglobulin-derived amyloidosis and to avoid treating other amyloidoses with potentially toxic chemotherapy. Immunofluorescence is the first step to type renal immunoglobulin-derived amyloidosis but the performance characteristics of this method are largely unknown. Here, we establish the sensitivity and specificity of immunofluorescence for diagnosing immunoglobulin-derived amyloidosis in patients whose amyloid typing was performed by the current gold standard of laser microdissection/mass spectrometry. Renal biopsy pathology reports originating from several institutions with a diagnosis of amyloidosis and which had amyloid typing by laser microdissection/mass spectrometry performed at our center were reviewed. Reported immunofluorescence staining for kappa or lambda of 2+ or more, with weak or no staining for the other light chain was considered positive for light chain amyloidosis by immunofluorescence. Based on microdissection/mass spectrometry results, of the 170 cases reviewed, 104 cases were typed as immunoglobulin-derived amyloidosis and 66 were typed as non-immunoglobulin-derived amyloidosis. Immunofluorescence sensitivity for diagnosing immunoglobulin-derived amyloidosis was 84.6%. The remaining 16 cases could not be diagnosed by immunofluorescence due to reported weak staining for all antigens or reported lack of preferential staining for one antigen. Immunofluorescence specificity was 92.4%. Five cases, all amyloid A amyloidosis, were misdiagnosed as immunoglobulin-derived amyloidosis by immunofluorescence. Immunofluorescence failed to accurately differentiate immunoglobulin-derived from non-immunoglobulin-derived amyloidosis in 12.3% of cases of renal amyloidosis. Relying on immunofluorescence alone for determining immunoglobulin-derived vs. non-immunoglobulin-derived amyloidosis may lead to misdiagnosis. Thus, immunofluorescence has inferior sensitivity and specificity compared with laser microdissection/mass spectrometry in the typing of immunoglobulin-derived amyloidosis.

Paraneoplastic Cast Nephropathy Associated With Pancreatic Mixed Acinar-Neuroendocrine Carcinoma: A Case Report.

Acute kidney injury is common in patients with cancer and may result from sepsis, obstruction, radiotherapy, chemotherapeutic agents, and nonsteroidal anti-inflammatory drugs. Rare reports of acute kidney injury due to cast nephropathy in patients with pancreatic acinar cell carcinoma have been described, but a pathogenetic link between cast nephropathy and carcinoma was not established. We report a patient with pancreatic mixed acinar-neuroendocrine carcinoma who developed severe acute kidney injury. Kidney biopsy showed cast nephropathy characterized by fractured periodic acid-Schiff-negative casts, associated with mononuclear and giant cell reaction. The patient did not have multiple myeloma and casts did not show immunoglobulin light chain restriction on immunofluorescence. Analysis using liquid chromatography-tandem mass spectrometry and immunohistochemistry identified 2 acinar cell-specific proteins, regenerating islet-derived 1α and carboxypeptidase A1, in both tubular casts and tumor cells. Thus, this case demonstrates that solid tumor-specific proteins can be nephropathic by obstructing renal tubules, resulting in acute kidney injury, a previously proposed but not characterized pathophysiologic mechanism for paraneoplastic nephropathy associated with carcinoma.

Clarifying immunoglobulin gene usage in systemic and localized immunoglobulin light-chain amyloidosis by mass spectrometry.

The goal of this study was to investigate the frequency of use of light-chain variable region (IGVL) genes among patients with systemic (ALS) and localized (ALL) amyloidosis and to assess for associations between IGVL gene usage and organ tropism. We evaluated clinic charts from 821 AL patients seen at the Mayo Clinic who had bone marrow, fat pad, and solid organ tissue samples typed by liquid chromatography tandem mass spectrometry (LC-MS). We identified 701 patients with ALS and 120 with ALL Overall, we were able to identify an IGVL gene in 87 (72%) patients with ALL and 573 (82%) patients with ALS When compared with ALL, LV6-57 was more common, whereas KV3-20 and heavy-chain codeposition were less common in ALS In this large series of ALS, characteristics particular to specific genotypes became apparent. LV6-57 patients were more likely to have renal involvement and to harbor a translocation 11;14. LV3-01 patients were less likely to have advanced cardiac disease and renal involvement. LV2-14 patients were more likely to have peripheral nerve involvement, an intact circulating immunoglobulin, and lower circulating dFLC. LV1-44 patients were more likely to have cardiac involvement. KV1-33 patients had more liver involvement and higher circulating dFLC. Finally, KV1-05 was associated with inferior overall survival but not independently of cardiac stage. IGVL gene usage appears to provide clues about disease pathophysiology and tissue tropism. LC-MS is a high-throughput and low-resource technique that can be used to identify IGVL gene from clinical tissue specimens.

DnaJ Heat Shock Protein Family B Member 9 Is a Novel Biomarker for Fibrillary GN.

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.

Congophilic Fibrillary Glomerulonephritis: A Case Series.

RATIONALE & OBJECTIVE: Congo Red positivity with birefringence under polarized light has traditionally permitted classification of organized glomerular deposits as from amyloid or nonamyloid diseases. The absence of congophilia has been used to differentiate fibrillary glomerulonephritis (GN) from amyloidosis. We describe a series of fibrillary GN cases in which the deposits are Congo Red-positive (congophilic fibrillary GN) and discuss the role of DNAJB9 in distinguishing congophilic fibrillary GN from amyloidosis. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Analysis of the clinicopathologic characteristics of 18 cases of congophilic fibrillary GN. Mass spectrometry was performed and compared with 24 cases of Congo Red-negative fibrillary GN, 145 cases of amyloidosis, and 12 apparently healthy individuals. DNAJB9 immunohistochemistry was obtained for a subset of cases. RESULTS: The proteomic signature of amyloid was not detected using mass spectrometry among cases of congophilic fibrillary GN. DNAJB9, a recently discovered proteomic marker for fibrillary GN, was detected using mass spectrometry in all cases of fibrillary GN regardless of congophilia and was absent in cases of amyloidosis and in healthy individuals. DNAJB9 immunohistochemistry confirmed the mass spectrometry findings. The congophilic fibrillary GN cases included 11 men and 7 women with a mean age at diagnosis of 65 years. Concomitant monoclonal gammopathy, hepatitis C virus infection, malignancy, or autoimmune disease was present in 35%, 22%, 17%, and 11% of patients, respectively. No patient had evidence of extrarenal amyloidosis. Patients presented with proteinuria (100%), nephrotic syndrome (47%), hematuria (78%), and chronic kidney disease (83%). After a mean follow-up of 23 months, 31% of patients progressed to end-stage kidney disease and the remaining 69% had persistently reduced kidney function. LIMITATIONS: Retrospective nature. Blinded pathology evaluations were not performed. CONCLUSIONS: The congophilic properties of organized fibrillary deposits should not be solely relied on in differentiating fibrillary GN from renal amyloidosis. Mass spectrometry and DNAJB9 immunohistochemistry can be useful in making this distinction.

Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a "wastebasket" category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Inferior survival in high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements is not associated with MYC/IG gene rearrangements.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (double-/triple-hit lymphoma) have an aggressive clinical course. We investigated the prognostic value of transformation from low-grade lymphoma, cytological features (high grade versus large cell), MYC rearrangement partners (immunoglobulin versus nonimmunoglobulin gene), and treatment. We evaluated 100 adults with double-/triple-hit lymphoma, reviewing cytological features; cell of origin; and rearrangements of MYC, BCL2, and BCL6 using MYC, BCL2, and BCL6 break-apart and IGH/MYC, IGL/MYC, IGK/MYC, and IGH/BCL2 dual-fusion interphase fluorescence in situ hybridization probes. Outcome analysis was restricted to patients with lymphoma, de novo or at transformation, who received anthracycline-based chemotherapy. Among them, 60% had high-grade cytological features; 91% had a germinal center B-cell phenotype, and 60% had a MYC/IG rearrangement. Germinal center B-cell phenotype was associated with BCL2 rearrangements (P<0.001). Mean (95% confidence interval) 5-year overall survival was 49% (37%-64%). Transformation from previously treated and untreated low-grade lymphoma was associated with inferior overall survival (hazard ratio, 2.99; P=0.008). Patients with high-grade cytological features showed a non-significant tendency to inferior outcome (hazard ratio, 2.32; P=0.09). No association was observed between MYC rearrangement partner and overall survival (hazard ratio, 1.00; P=0.99). Compared with patients receiving rituximab, cyclophosphamide, doxorubicin, and vincristine (R-CHOP) and dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R), patients receiving rituximab, cyclophosphamide, vincristine, doxorubicin, methotrexate/ifosfamide, etoposide, and cytarabine (R-CODOX-M/IVAC) had a non-significant tendency to better overall survival (hazard ratio, 0.37; P=0.10). In conclusion, high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements had heterogeneous outcomes and MYC/IG rearrangements were not associated with inferior overall survival.

Spheroid-type of AL amyloid deposition associated with colonic adenocarcinoma: A case report with literature review.

We report a colonic adenocarcinoma associated with diffuse submucosal deposition of a peculiar spheroid-type amyloid identified in the colon, terminal ileum, and appendix. A 65-year-old woman with past medical histories of hypertension, and chronic obstructive pulmonary disease, presented to the emergency room with cramping abdominal pain and nausea. A computed tomography (CT) scan of abdomen showed right colonic volvulus. Emergency right hemicolectomy was performed. The specimen showed colonic adenocarcinoma with focal submucosal invasion (pT1) arising from a villotubular adenoma. A diffuse submucosal spheroid-type amyloid deposition (resembling corpora amylacea-like structures with Liesegang ring formation) was identified in the colon, ileum, and appendix. Electron microscopy examination of this unusual spheroidal-type material further confirmed the presence of amyloid fibrils. Analysis by liquid chromatography-mass spectrometry detected AL (lambda) type amyloidosis in this specimen. Tests for monoclonal gammopathy were not performed because patient consent was not obtained. In tissue section evaluation, however, no plasma cell neoplasm was identified. Cases with isolated AL amyloid deposition in the gastrointestinal tract have been reported rarely, and there is no case report of colonic adenocarcinoma associated with primary amyloid deposition in the English literature.

Hereditary Lysozyme Amyloidosis Variant p.Leu102Ser Associates with Unique Phenotype.

Lysozyme amyloidosis (ALys) is a rare form of hereditary amyloidosis that typically manifests with renal impairment, gastrointestinal (GI) symptoms, and sicca syndrome, whereas cardiac involvement is exceedingly rare and neuropathy has not been reported. Here, we describe a 40-year-old man with renal impairment, cardiac and GI symptoms, and peripheral neuropathy. Renal biopsy specimen analysis revealed amyloidosis with extensive involvement of glomeruli, vessels, and medulla. Amyloid was also detected in the GI tract. Echocardiographic and electrocardiographic findings were consistent with cardiac involvement. Proteomic analysis of Congo red-positive renal and GI amyloid deposits detected abundant lysozyme C protein. DNA sequencing of the lysozyme gene in the patient and his mother detected a heterozygous c.305T>C alteration in exon 3, which causes a leucine to serine substitution at codon 102 (Human Genome Variation Society nomenclature: p.Leu102Ser; legacy designation: L84S). We also detected the mutant peptide in the proband's renal and GI amyloid deposits. PolyPhen analysis predicted that the mutation damages the encoded protein. Molecular dynamics simulations suggested that the pathogenesis of ALys p.Leu102Ser is mediated by shifting the position of the central ß-hairpin coordinated with an antiparallel motion of the C-terminal helix, which may alter the native-state structural ensemble of the molecule, leading to aggregation-prone intermediates.

Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII.

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.

Clinical, biopsy, and mass spectrometry findings of renal gelsolin amyloidosis.

Gelsolin amyloidosis is a rare type of amyloidosis typically involving the cranial and peripheral nerves, but rarely the kidney. Here we report the clinical, kidney biopsy, and mass spectrometry findings in 12 cases of renal gelsolin amyloidosis. Of the 12 patients, five were men and seven were women with mean age at diagnosis of 63.8 years. Gelsolin amyloidosis was most common in Caucasians (six patients) and Asians (four patients), and included one each African-American and Hispanic patients. Nephrotic syndrome was the most common cause of biopsy, although most patients also had progressive loss of kidney function. Hematological and serological evaluation was negative in 11 patients, while one patient had a monoclonal gammopathy. The renal biopsy showed large amounts of pale eosinophilic Congo red-positive amyloid deposits typically restricted to the glomeruli. Immunofluorescence studies were negative for immunoglobulins in nine cases with three cases of smudgy glomerular staining for IgG. Electron microscopy showed mostly random arrangement of amyloid fibrils with focally parallel bundles/sheets of amyloid fibrils present. Laser microdissection of the amyloid deposits followed by mass spectrometry showed large spectra numbers for gelsolin, serum amyloid P component, and apolipoproteins E and AIV. Furthermore, the p. Asn211Lys gelsolin mutation on mass spectrometry studies was detected in three patients by mass spectrometry, which appears to represent a renal-limited form of gelsolin amyloidosis. Thus, renal gelsolin amyloidosis is seen in older patients, presents with nephrotic syndrome and progressive chronic kidney disease, and histologically exhibits glomerular involvement. The diagnosis can be confirmed by mass spectrometry studies.

Characterization of C3 in C3 glomerulopathy.

Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3ß), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3ß that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3ß. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3ß amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.

Long-term follow-up of chemoimmunotherapy with rituximab, oxaliplatin, cytosine arabinoside, dexamethasone (ROAD) in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma: Results of a study of the Mayo Clinic Cancer Center Research Consortium (MCCRC) MC0485 now known as academic and community cancer research united (ACCRU).

Patients with relapsed aggressive non-Hodgkin lymphoma (NHL) are often treated with platinum-based chemoimmunotherapy regimens in preparation for autologous stem cell transplant. We sought to reduce toxicity and maintain efficacy by using oxaliplatin with rituximab, cytarabine and dexamethasone (ROAD) in a phase II clinical trial in patients who had relapsed after one prior regimen. ROAD was delivered q21 days and consisted of rituximab 375 mg/m2 IV weekly x 4 doses (cycle 1 only); dexamethasone 40 mg PO/IV d2 - 5; oxaliplatin 130 mg/m2 IV day 2; cytarabine 2000 mg/m2 IV × two doses on days 2 to 3; and pegfilgrastim 6 mg SC on day 4. Forty-five eligible patients were accrued between 2006 and 2008. Patient characteristics were a median age of 69 years; 96% had received prior rituximab; 53% were within one year of diagnosis. The median number of cycles received was 2 (range, 1-6). Forty-four % received ROAD as an outpatient. The overall response rate was 71% with 27% (12/45) CR and 44% (20/45) PR. Forty-four % (20/45) of all patients and 69% (18/26) of patients whom responded after 2 cycles proceeded to transplant. Median overall survival was 26 mos (95% CI: 7.3 mos-not reached) and median progression-free survival was 11 mos (95% CI: 6-104 mos). There was no grade 3/4 nephrotoxicity; the rate of grade 3/4 neuropathy was 4%. Forty-two percent of all patients and 69% of patients transplanted remain alive at 5 years. ROAD represents an acceptable salvage therapeutic option for patients with relapsed aggressive NHL.

Axillary Intraneural Ganglion Cysts.

Aside from affecting the stability of the glenohumeral joint, tears in the joint capsule can give rise to extraneural (paralabral) and, very rarely, intraneural ganglion cysts. This report presents the first two cases of axillary intraneural ganglion cysts in the literature with magnetic resonance imaging. Both cases were incidentally noted to have coexisting lesions (lymphadenopathy from an undifferentiated malignancy and suprascapular nerve entrapment, respectively). This report reinforces the applicability of the articular theory for intraneural ganglion cysts at a novel site.

Clinical, biopsy, and mass spectrometry characteristics of renal apolipoprotein A-IV amyloidosis.

Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis with only a single reported case. Here we describe the clinical, biopsy, and mass spectrometry characteristics of 11 cases of renal AApoAIV amyloidosis encompassing 9 men and 2 women with a mean age at diagnosis of 63.5 years. Progressive chronic kidney disease (mean serum creatinine 2.9 mg/dl) was the most common cause for biopsy with proteinuria absent or minimal in all except one. Hematological and serological evaluation was negative in 9 patients, while 2 had a monoclonal gammopathy. The renal biopsy findings were striking and showed large amounts of eosinophilic Congo-red positive amyloid deposits restricted to the renal medulla with sparing of the renal cortex. In 6 cases, peritubular amyloid was noted in addition to the interstitial involvement. Immunofluorescence studies were negative for immunoglobulins. Electron microscopy showed nonbranching fibrils measuring 7 to 10 nm in diameter. Laser microdissection of the amyloid deposits followed by mass spectrometry showed large spectra number (a semiquantitative measure of abundance) for AApoAIV protein ranging from 49 to 169 (average 85), serum amyloid protein (average 19), and apolipoprotein E (average 48). Importantly, no peptides were detected for any other forms of known amyloidogenic precursor proteins. Thus, renal AApoAIV amyloidosis typically presents with progressive chronic kidney disease and histologically exhibits extensive medullary involvement with sparing of the cortex. The diagnosis is best established by mass spectrometry. Hence, a high degree of suspicion and examination of the renal medulla is required to make the diagnosis.

Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a common form of renal amyloidosis among Egyptians.

Large case series of renal amyloidosis subtypes have recently been published in the United States and Europe showing AL amyloidosis to be the predominant subtype in this part of the world. However, the most common subtypes of renal amyloidosis throughout the rest of the world are unknown. We present here the first large case series detailing the subtypes of renal amyloidosis among Egyptians. In this population, AA amyloidosis was the most common type of amyloidosis on renal biopsy at 48%. The newly described leukocyte chemotactic factor 2 amyloidosis (ALECT2) was the second most common type and represented nearly one-third of renal amyloid cases at 31%. AL accounted for only 20% of cases. The pathologic findings in ALECT2 cases were similar to those previously described in other case series. Thus ALECT2, which was initially thought to affect mainly Hispanics in the United States, appears to represent an important and likely underrecognized etiology of chronic kidney disease among Egyptians and probably in other ethnic groups around the world.