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Abstract Background: Amyloidosis caused by immunoglobulin light chain (IGLC) deposition, so-called AL-type or primary amyloidosis, is the most common type of amyloidosis. It has been long believed that IGLC variable regions form the core of the AL-type amyloid deposits and peptides derived from IGLC constant region peptides are only occasionally integrated into this core. For this reason, the scientific effort to identify thge risk factors for development of AL amyloidosis and the biochemical characteristics amyloid deposits has focused on IGLC variable region derived proteins. To understand the peptide constituents of AL amyloidosis better, we undertook a comprehensive study of AL amyloidosis using a novel mass spectrometry based proteomic analysis approach. Methods: Paraffin embedded tissue from 100 cases of AL amyloidosis was studied. In each case amyloid type was previously established by clinical and pathological examination. Congo red stained paraffin sections were prepared and amyloid deposits were microdissected by laser microdissection microscopy. The microdissected tissue fragments were processed and trypsin digested into peptides. The peptides were analyzed by nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS). The resulting LC-MS/MS data were correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Results and Discussion: LC-MS/MS gave peptide profiles consistent with AL amyloidosis in each case. The analysis showed IGLC-lambda deposition in 66 cases and IGLC-kappa deposition in 34 of cases. In each case, LC MS/MS confirmed the previous clinicopathological diagnosis. Interestingly peptides representing IGLC constant region were present in each case. Using this LC-MS/MS methodology, theoretically it is possible to cover 78% of the IGLC-lambda and 87% IGLC-kappa constant regions. In our samples, the average coverage of the IGLC-lambda and IGLC-kappa constant regions were 40% (range 14–78%)and 55% (range 16–87%) respectively. Additionally, the distribution of the peptides suggested that in the majority of the cases whole of the IGLC constant region was deposited. LC MS/MS also identified IGLC-lambda variable region peptides in 37 of 66 cases and IGLC-kappa variable region peptides in 29 of 34 cases studied. The variable region coverage was more restricted and the peptides identified were frequently within the framework segments. It is likely that the peptides derived from CDR segments were present but not detected by the methodology as somatic hypermutation randomly alters the amino acid sequence in the CDR segments and such new sequences are not available in public databases used by algorithms for peptide identification. In the cases with the IGLC variable region hits, it was also possible to assign variable region family usage. IGLC-lambda cases frequently used IGLC-lambda variable region I, II and III families whereas, in IGLC-kappa cases, IGLC-kappa variable region I and III families dominated. Conclusions: AL amyloidosis can be accurately diagnosed using laser microdissection and LC-MS/MS based proteomic analysis in routine clinical specimens. AL amyloidosis invariably contains IGLC constant region peptides and, frequently, the whole of the constant region is deposited. This finding suggests that studies on molecular pathogenesis of amyloidosis should not only consider the IGLC-variable region but also the constant region. It is possible to identify IGLC variable region family usage in AL amyloidosis using LC MS/MS based proteomic analysis. In the clinical setting, this information may be helpful in predicting organ distribution and clinical outcome.

Abstract The management of systemic amyloidosis relies on the treatment of the underlying etiology and differs radically for different amyloid types. Therefore, given that at least 25 different proteins have been associated with amyloidosis, accurate identification of proteins deposited as amyloid fibrils is an important clinical problem. In this study, we describe a novel method that can characterize amyloid subtypes using laser microdissection (LMD) and mass spectrometry (MS) on routinely processed paraffin-embedded tissues. The study used 60 cases consisting of 16 transthyretin, 9 serum amyloid-associated protein, 20 immunoglobulin light chain lambda, 5 immunoglobulin light chain kappa, and 10 amyloid negative control samples. The biopsy specimens studied included heart, kidney, gastrointestinal tract, lung and decalcified bone marrow specimens. The amyloid type in all cases was previously characterized based on clinical findings, immunohistochemistry and, where indicated, by molecular testing for transthyretin mutations. Amyloid plaques were captured from an 10 micron paraffin section exhibiting positive Congo Red staining using LMD. Proteins were extracted, digested with trypsin and identified following MS/MS using the Mascot search algorithm analysis. MS correctly identified each of the 4 types of amyloidosis analyzed. Serum Amyloid P component and Apolipoprotein E were also identified as constituents of the amyloid deposition. The analysis was successful on all tissue types including decalcified bone marrow specimens and small biopsy specimens such as endomycardial biopsies and renal biopsies. The use of LMD from paraffin embedded biopsies and subsequent analysis by MS allows identification of the type of amyloid protein deposited with high specificity and sensitivity. This method promises to be a clinical test for accurate identification of amyloid proteins in routinely processed biopsy specimens and overcomes many of the specificity and sensitivity issues associated with current methods such as immunohistochemistry.

Abstract The amyloidoses are heterogeneous diseases associated with the deposition of insoluble proteins or peptides with a characteristic beta-diffraction pattern extracellularly. At the current time, over 27 different extracellular fibril proteins are known to cause disease in humans. Iatrogenic amyloidosis is a rare, often not sought diagnosis. On occasion, patients with drug induced amyloidosis can present with other systemic features reminiscent of systemic immunoglobulin-derived (AL) amyloidosis, and may present a diagnostic challenge. Treatment of the latter often involves chemotherapy and/or stem cell transplantation, while the former tends to remain localized and needs local therapy; thus accurate diagnosis is critical. To this end, proteomic analysis of amyloid tissue has proven to be an invaluable tool in amyloid typing. We have analyzed the biochemical composition of iatrogenic amyloid using laser capture/tandem mass spectrometry (LC-MS/MS)-based proteomic analysis in 52 cases of insulin and enfuvirtide (Fuzeon¨) associated amyloidosis. In brief, 10-μm-thick sections of formalin-fixed paraffin-embedded tissues were stained with Congo red. Congo red positively staining tissue as viewed with a fluorescent light source appeared bright red. Positive areas were dissected using laser microdissection to a volume of at least 60,000 μm2; three microdissections were analyzed for each case. The microdissected material was collected into 0.5-ml microcentrifuge tube caps containing 35 μL Tris/EDTA/0.002% Zwittergent buffer. Microdissected fragments were subjected to a heat-mediated antigen retrieval method (98C for 90minutes) before being denatured via sonication and subsequently digested into tryptic peptides overnight using 0.5ug of trypsin. The resulting digests were then analyzed with nanoflow LC-MS/MS. The MS/MS spectra of each case were matched against a composite protein sequence database using three different search algorithms (Sequest, X!Tandem, and Mascot). The composite database contained the human SwissProt entries but was also augmented with known immunoglobulin variant domains, known amyloidogenic mutations from literature, the enfuvirtide amino acid sequence, and common contaminants. Reversed protein sequences were appended to the database for estimating the false discovery rates of the identifications. The peptide identification results were filtered using Scaffold software (Proteome Software, Portland, OR) and then filtered peptides were assembled into protein identifications. Candidate proteins with at least one high-confident (probability of identification >90%) unique peptide identification and at least four MS/MS spectral matches were considered for clinical interpretation. For each case, we created a personalized proteomic profile that lists all the confident protein identifications in each of the microdissection along with their respective MS/MS spectral counts. The number of MS/MS spectra matching to a protein is considered as a semi-quantitative measure of its abundance. The most abundant amyloidogenic protein detected across all microdissections and as interpreted in the context of the clinical history is considered to be the amyloid subtype. Figure 1 shows the results of insulin amyloidosis. Figure 2 shows the results of enfuvirtide amyloidosis. Amyloid deposits are shown to be composed of the recombinant drug in addition to amyloid precursor proteins such as apolipoprotein A-I, A-IV, E and serum amyloid P (SAP).Figure 1. Insulin-associated amyloidosisFigure 1. Insulin-associated amyloidosisFigure 2Enfuvirtide (Fuzeon¨)-associated amyloidosisFigure 2. Enfuvirtide (Fuzeon¨)-associated amyloidosis In conclusion, we show the biochemical composition of all known drug-induced iatrogenic amyloidosis and provide the utility of proteomic analysis in elucidating amyloid subtyping for accurate diagnosis and management. Legend: A spectral count number of greater than 4 is significant. The green boxes denote protein identification at a probability of over 95%, and yellow 80-94%. In figure 1, insulin (# 2) and in figure 2, enfuvirtide (#5) are shown in abundance, additionally other amyloid precursors such as apolipoproteins A-IV, E, A-I and SAP are also seen. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Immunoglobulin light chain-associated amyloidosis (AL) is caused by deposition of immunoglobulin light chain molecules with unique, clonotypic variable (V) regions. Detection and identification of the V region, however, has been challenging due heterogeneity inherent in V regions. We developed a new method for detecting IGKV and IGLV region fragments in AL deposits using protein mass spectrometry and novel bioinformatics approach. We report distinct IGKV and IGLV usage in localized versus systemic AL. Methods Shotgun protein mass spectrometry on amyloid plaques was performed as previously described (Blood. 2009; 114: 4957-9). Peptide tandem mass spectra (MS/MS) were searched against a composite sequence database containing SwissProt's human complete proteome augmented with 1764 IG V region sequences obtained from ImMunoGeneTics database and a Mayo Clinic internal database. Reversed sequences were appended to the database for estimating identification false discovery rates (FDRs). Peptide identifications were processed with Scaffold software. Confident protein identifications (probability > 0.9) with at least one unique peptide identifications and five MS/MS matches were considered. Detected variable gene family (if any) with most number of peptide and spectral matches is considered to be present in the deposit. The method was first validated in 8 cases of AL amyloidosis with known IGKV and IGLV region sequences and then applied on 1238 systemic and 393 localized AL cases. Differences between groups were measured by generating hypergeometric p-values. Results In all 8 AL cases with known IGKV and IGLV region usage, gene family identified in the AL deposit via proteomics matched the gene family inferred from bone marrow plasma cells by Sanger sequencing of IGLC genes. Armed with this method, we analyzed the amyloid proteomics data from 1238 patients with systemic AL amyloidosis and 393 patients with localized AL amyloidosis. The anatomical site distribution in systemic cohort was 296 GI tract, 364 heart, 225 kidney, 81 liver and 272 fat aspirate; Localized cohort has 78 bladder, 158 lung and 157 skin cases. Figure 1 shows the normalized frequency of the variable gene families detected between the systemic and localized AL amyloidosis. For AL-kappa, KV1 was more prevalent in systemic cases when compared to localized cases (p=7.2E-12) suggesting KV1 clones as a hallmark for systemic AL-kappa amyloidosis. KV3 was more frequently seen in localized AL-kappa cases when compared to systemic AL-kappa cases (p =2.8E-11). For AL-lambda, LV1 and LV2 gene families were more prevalent in localized cases when compared to systemic cases (p= 0.039 and 2.7E-05). LV6 was more prevalent in systemic AL-lambda cases (p=1.4E-07). We also detected a higher incidence of mixed AL/AH type in localized AL (18%) when compared to systemic AL (5%; Odds Ratio=4.2673, p<0.0001). We next turned to the organ specific IGKV and IGLV gene family usage patterns in patients with localized amyloidosis. For 147 localized AL-kappa cases, KV3 was most dominant in lung (64%) and KV1 was dominant in skin (38%). For 246 localized AL-lambda cases, LV2 was most prevalent in lung (34%) and LV3 was most prevalent in skin (36%). For bladder, both LV1 and LV2 had comparable prevalence (42% and 32%). For 361 systemic AL-kappa cases, KV1 consistently ranked at the top of gene families used in GI tract (55%), heart (71%), kidney (59%), liver (75%) and fat aspirate (50%). For 877 systemic AL-lambda cases, LV2 was predominant in GI tract (30%), LV3 in heart (35%) and LV6 in kidney (33%). The prevalence of LV1, LV2 and LV3 in liver is comparable (36%, 22% and 31%). LV6 and LV3 had comparable prevalence (22% and 24%) in AL-lambda fat aspirates. Conclusion The novel proteomics method detects IGLC V family usage in large cohorts of AL patients. It identifies unique profiles in systemic and localized cases, and in different organ sites. This information will be helpful in determining systemic versus localized nature of AL amyloidosis at diagnosis and to assess risk of specific end organ involvement. We found also a strong association between IGKV and IGLV gene usage and organ involvement. Disclosures: No relevant conflicts of interest to declare.

Abstract Amyloidosis is a group of hereditary and acquired disorders of diverse pathogenesis united by end organ damage due to tissue deposition of misfolded proteins. The diagnosis is typically based on demonstrating Congo red positive amyloid deposits in tissue biopsies of clinically involved sites. Because treatment options for amyloidosis patients are highly varied and intricately tied to the identity of the protein that constitutes the amyloid deposits, adequate diagnostic evaluation now requires identification of the amyloidogenic protein. Traditionally, immunohistochemistry with antibodies to amyloid-associated proteins was the clinical technique of choice for subtyping amyloid deposits in tissue sections. This technique suffers from poor analytical sensitivity and specificity and is a suboptimal technique on which to base treatment decisions for amyloidosis patients. We developed a highly accurate and reproducible liquid chromatography electrospray tandem mass spectrometery (LC-MS/MS)-based shotgun proteomics assay for subtyping amyloid deposits (Blood. 2009; 114: 4957-9) from microdissected paraffin embedded tissue samples. Implementation of this assay in a CAP/CLIA clinical laboratory has subsequently transformed the standard of amyloidosis care at Mayo Clinic. Since 2008, we utilized the assay in routine practice to identify 20 different amyloid types in tissue samples from diverse anatomic sites from 4139 patients (Table 1). Although AL and ATTR were most common, the cases include a large variety of unusual, and in many cases clinically unsuspected amyloid types. In addition, the amyloid protein subtypes had different frequencies in different anatomic sites (A partial list of tissue sites and their amyloid types analyzed in this study includes: bone marrow: AL 82%, ATTR 15%, AA 2%, other 1%; kidney: AL 51%, ALect2 18%, AA 17%, AH 5%, AFib 4%, other 5%; liver: AL 64%, ALect2 23%, AApoA1 6%, AA 3%, ATTR 2%, other 2%; heart: ATTR 60%, AL 37%, AANF1%, other 2%; GI tract: AL 78%, ATTR 13%, AA 6% AApoA4 1%, other 2%; eye [primarily adnexae] AL 63%; eye [primarily cornea] TGFB induced protein ig-h3 34%, other 3%). One caveat is that these distributions of amyloid types may not accurately reflect population prevalence. More likely, the findings reflected a composite prevalence that included challenging amyloidosis cases.Table 1Frequency of amyloid subtypes observed in the Mayo Amyloid Cohort (n=4139). Asterisk denotes non-canonical subtypesAmyloid SubtypeNumber (%) of casesAL2553 (61.68)ATTR1015 (24.52)AA151 (3.65)ALect2148 (3.58)AIns48 (1.09)Keratin*36 (0.87)AApoA130 (0.72)AH27 (0.65)AFib26 (0.63)TGFB1-IP*22 (0.53)AApoA420 (0.48)AANF14 (0.34)Ab2M12 (0.29)AGel12 (0.29)ASem112 (0.29)APro7 (0.17)ALys3 (0.07)ACal2 (0.05)Enfuvirtide*2 (0.05)AIAPP2 (0.05) The prevalence of diverse and unexpected amyloid types in many tissues suggest that shotgun proteomic-based analysis may be the only way to accurately assign amyloid subtype in tissue samples. The technique avoids the analytical problems of immunohistochemistry and the bias introduced by assigning probable amyloid type based on clinical presentation and disease distribution. It provides the clinician with an amyloid subtype that allows for confident individualized patient management. Accurate amyloid subtyping by LC-MS/MS is not only possible, but has proven to be a robust routine clinical tool for the evaluation of numerous amyloidosis patients in our practice. Disclosures: No relevant conflicts of interest to declare.

Abstract In multiple myeloma, the neoplastic plasma cells are either dispersed throughout the red marrow spaces of the axial and proximal appendicular skeleton or concentrated in discrete, well vascularlized plasmacytomas that stimulate osteoclastic bone resorption and erode into cortical bone. Interactions between bone marrow stromal cells (BMSC) and myeloma cells are clearly important in the pathogenesis of myeloma that diffusely infiltrates the bone marrow and where plasmacytomas are in contact with the bone marrow space. However, these interactions cannot be of major relevance at the centers of expanding plasmacytomas since these sites are devoid of osteoclasts, osteoblasts and other BMSC. Histological sections of skeletal plasmacytomas from 22 patients with multiple myeloma were therefore examined after immunostaining with anti-macrophage antibodies. Hematoxylin and eosin stained sections showed sheets of plasma cells interspersed with numerous small blood vessels. Immunostained sections revealed abundant CD68+, CD163+ macrophage infiltrates in all tumors, and scattered collections of CD3+ T lymphocytes. Tumor-associated macrophages (TAM) accounted for 2 to 12 percent of nucleated cells in these plasmacytomas and were evenly distributed through the parenchyma or clustered around CD34+ blood vessels. In general the TAM had dendritic morphology, and each dendrite was in close contact with multiple plasma cells. In a small percentage of cases, there was a subpopulation of TAM strikingly clustered around CD34+ positive blood vessels. However, vascular mimicry was not seen. Abundant murine CD68+ TAM were also found in subcutaneous KAS6/1 and MM1 myeloma xenografts in SCID mice. To determine whether cells of the monocytic lineage might be exploitable as carriers for systemic delivery of therapeutic agents to plasmacytomas, monocyte-derived dendritic cells were infected with oncolytic measles viruses and administered intravenously to SCID mice bearing subcutaneous KAS6/1 xenografts. Immunostaining for human MHC II antigen revealed that the infused DCs could localize to the KAS6/1 tumors where they heterofused with and transferred MV infection to the myeloma cells. This is the first report of TAM in skeletal plasmacytomas of myeloma patients. Further research into the role of TAM in supporting myeloma growth is warranted since they may offer many potential opportunities for the development of novel approaches to myeloma therapy.

Abstract Abstract 1909 AL amyloidosis is the most common type of systemic amyloidosis. It is caused by extracellular deposition of circulating immunoglobulin light chains (LC) thought to be secreted by a primarily bone marrow based clonal plasma cell proliferative disorder. Although, invariably, the type of LC secreted by the plasma cell clone is identical to the LC deposited in the amyloid plaque, a comprehensive analysis of the re-arranged clonal LC gene coding secreted LC protein, the protein sequence of the serum and urine monoclonal band and the LC protein deposited in the amyloid plaques have not been previously performed. In this study, we compared the protein sequence predicted by the clonal LC gene sequence with the protein sequence identified in the serum and urine monoclonal bands and protein plaques by mass spectrometry (MS) based proteomics. 10 cases with systemic AL amyloidosis were studied. Bone marrow samples containing the clonal plasma cells were obtained and clonal LC variable gene sequences were determined by PCR amplification, cloning and sequencing as described previously (Abraham et al, Blood 2003, 101: 3801). For protein analysis, monoclonal protein bands identified in serum and/or urine protein electrophoresis and immunofixation were isolated from archival gels. For amyloid plaque protein analysis, the plaques were microdissected from paraffin embedded tissue. To identify the protein amino acid sequence of serum and urine bands and amyloid plaques, the samples were digested into peptides by trypsin and analyzed by MS based proteomic analysis. (see Vrana et al. and Blood 2009; 114: 4957 for methods). The results for amino acid sequence predicted from the clonal LC gene sequence, determined directly by MS from the serum and/or urine monoclonal band and amyloid plaques were then aligned and compared. In all 10 cases, the LC amino acid sequences predicted by sequencing of the clonal LC gene from bone marrow plasma cells, the amino acid sequence obtained directly from the monoclonal serum and/or urine protein and the amino acid sequence obtained from the amyloid plaque were identical. In most cases, both the complete variable region and constant regions were deposited as amyloid plaques. Some amyloid plaques showed cleavage of first 16 amino acids of the variable region suggesting that this part was not required for amyloid deposition. All LC variable region gene sequences were affected by somatic hypermutation, and identical mutations were present in both the serum and urine bands and in the amyloid plaques. In conclusion, these results, for the first time, comprehensively show that the monoclonal protein secreted by the neoplastic plasma cells is the pathogenetic protein causing the amyloid deposits in AL amyloidosis. Amyloid deposits contain virtually all of the LC protein identified in the serum or urine including both variable and constant regions, suggesting that the presence of the constant region may also play a role in amyloidogenesis. The technological capability to determine the complete protein sequence of the amyloidogenic monoclonal serum and urine LC protein isolated from archived gels will create new opportunities to develop diagnostic, predictive and prognostic serum or urine based tests for amyloidosis. Disclosures: Theis: Mayo Clinic: Patents & Royalties. Vrana:Mayo Clinic: Patents & Royalties. Dogan:Mayo Clinic: Patents & Royalties.

Abstract Peripheral T- and NK-cell lymphomas (PTCLs) are fatal in the majority of patients, and conventional chemotherapy is minimally effective. Targeted inhibition of tyrosine kinases (TKs) might be a useful treatment strategy, but TK overexpression by PTCLs has not been well characterized (except ALK). Recently a novel translocation t(5;9) fusing the ITK and SYK genes was reported in a subset of unspecified-type PTCLs, associated with high SYK gene expression and Syk protein positivity. Syk is a non-receptor TK important in immunoreceptor signal transduction, providing proliferation and survival signals in a variety of hematopoietic cells. Syk is absent or expressed at very low levels in most normal post-thymic T cells, where this signal transduction role is fulfilled by ZAP70. Since Syk TK inhibitors are now in clinical trials for other diseases, we sought to determine the incidence of t(5;9) and Syk overexpression in PTCL. Tissue microarrays were constructed using triplicate cores from paraffin-embedded tissue blocks. Cases were studied using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Additional studies were performed using flow cytometry and Western blot. There were 141 PTCLs, including 35 angioimmunoblastic, 66 unspecified, 21 anaplastic large cell, 2 enteropathy-associated, 5 hepatosplenic, 5 extranodal NK/T, and 7 other. No evidence of t(5;9) was identified by FISH in 86 informative cases. Normal T-cell subsets from peripheral blood, spleen, and lymph node were negative for Syk by IHC, flow cytometry, and Western blot. Syk was positive by immunohistochemistry in 133 of 141 (94%) PTCLs. All 8 Syk negative cases were extranodal, cytotoxic PTCLs. Western blot on frozen tissue correlated with IHC results in paraffin. All 4 Syk-positive cases tested showed phosphorylation of tyrosine residues 525/526, known to be required for Syk activation. We conclude that Syk is overexpressed in most PTCLs, including the vast majority of nodal cases. Syk may substitute for ZAP70 in these tumors and provide ongoing proliferation and survival signaling. The overexpression of Syk and phosphorylation of its Y525/526 residues suggest Syk may be a suitable target for pharmacologic TK inhibition as a treatment option for patients with PTCL.

Abstract Abstract 3992 Little is known about the rare entities of heavy and light chain amyloidosis (AHL) and heavy chain amyloidosis (AH). In this study, we report the renal and hematologic characteristics, pathology, and outcome of 17 patients with renal AH/AHL including 5 with AH (4 IgG and 1 IgA) and 12 with AHL (7 IgGλ, 3 IgAκ, 1 IgAλ, and 1 IgMλ), and compare them with 202 patients with renal AL amyloidosis (AL) diagnosed during the same time period. All cases were diagnosed by kidney biopsy that showed Congo red-positive deposits. Amyloid typing was done by laser microdissection and mass spectrometry (LMD/MS) (12 patients) or by immunofluorescence (5 patients). All patients with renal AH/AHL were Caucasians, with a M:F ratio of 2.4 and a median age at biopsy of 63 years. Compared with patients with renal AL, those with renal AH/AHL had less frequent concurrent cardiac involvement, higher likelihood of having circulating complete monoclonal Ig, lower sensitivity of fat pad biopsy and bone marrow biopsy for detecting amyloid, higher incidence of hematuria, and better patient survival. The hematologic and renal responses to chemotherapy were comparable to renal AL. In 42% of patients, AH/AHL could not have been diagnosed without LMD/MS. In conclusion, renal AH/AHL is an uncommon but under-recognized form of amyloidosis, and its diagnosis is greatly enhanced by the use of LMD/MS for amyloid typing. The accurate histological diagnosis of renal AH/AHL and distinction from AL may have important clinical and prognostic implications. Table 1. Demographics and hematologic characteristics AH/AHL AL p value No. of patients 17 202 Gender: Male/female 12/5 (71%/29%) 126/76 (62%/38%) 0.61 Age, median (range) 63 (50–77) 62 (36–86) 0.73 Additional organ involvement 8 (47%) 126 (62%) 0.3 Cardiac involvement 3 (18%) 100 (50%) 0.01* % of plasma cells in bone marrow, median (IQR) 8 (5–15) 6 (5–10) 0.82 ≥30 plasma cells 4 (24%) 11/198 (6%) 0.02* Positive SPEP/SIF for paraprotein 15 (88%) 158/200 (79%) 0.53 Presence of whole monoclonal protein on SPEP 14 (82%) 108/200 (54%) 0.04* Positive UPEP/UIF for paraprotein 13/16 (81%) 158/189 (84%) 0.73 Presence of whole monoclonal protein on UPEP 10/16 (63%) 61/189 (32%) 0.03* Abnormal serum FLC ratio (<0.26 or >1.65) 9/12 (75%) 150/188 (80%) 0.71 Markedly abnormal FLC ratio (< 0.125 or > 8) 5/12 (42%) 100/188 (53%) 0.55 Positive bone marrow for amyloid 6/16 (38%) 135/183 (74%) 0.004* Positive fat pad biopsy for amyloid 2/14 (14%) 105/145 (72%) <0.001* IQR, interquartile range. Table 2. Renal characteristics at kidney biopsy AH/AHL AL p value No. of patients 17 202 24h urine protein in g, median (IQR) 5.1 (3.2–9.0) 6.0 (3.2–10.0) 0.9 Full nephrotic syndrome 9/16 (56%) 132/197 (67%) 0.42 Serum albumin in g/dl, median (IQR) 2.7 (2.2–3.3) 2.5 (1.9–2.9) 0.29 % albuminuria on UPEP, median (IQR) 68 (61–72) 70 (60–76) 0.47 Serum creatinine in mg/dl, median (IQR) 1.4 (1.1–2.1) 1.2 (0.9–1.8) 0.25 Serum creatinine >1.2 mg/dl 10/16 (63%) 92/201 (46%) 0.3 eGFR, median (IQR) 47 (27–67) 58 (36–75) 0.29 Decreased eGFR 10/16 (63%) 103/201 (51%) 0.44 Microscopic hematuria 9/16 (56%) 44/169 (26%) 0.02* IQR, interquartile range. Disclosures: No relevant conflicts of interest to declare.

The 2017 World Health Organization update introduced a new category of nodal peripheral T-cell lymphoma with T-follicular helper phenotype (PTCL-TFH) defined by expression of at least 2 or 3 TFH markers. Our study assesses the utility of an immunohistochemical panel of 5 TFH markers (CD10, BCL6, PD-1, CXCL13, and ICOS) for identification of TFH phenotype in angioimmunoblastic T-cell lymphoma (AITL) and PTCL not otherwise specified (NOS). Immunohistochemical for the 5 markers was performed on 22 cases of AITL and 29 cases of PTCL-NOS. Cases were reviewed for morphologic features characteristic of AITL. All AITL cases showed expression of ≥2 TFH markers. This panel resulted in reclassification of 41% PTCL-NOS cases to PTCL-TFH. Positive percent agreement for the TFH phenotype is 97% for PD1, 94% for ICOS, 44% for CD10 and CXCL13, and 29% for BCL6. Negative percent agreement for TFH phenotype is 100% for CD10, BCL6, and CXCL13, 82% for ICOS and 71% for PD1. AITL cases were more likely than PTCL-TFH cases to contain expanded CD21-positive follicular dendritic cell meshworks, clear cell cytology and polymorphous inflammatory background; however, there was a significant (P<0.005) Kruskal-Wallis trend in all morphologic variables between the 3 groups suggesting a continuum from PTCL-NOS to PTCL-TFH to AITL. The median number of morphologic features of AITL also correlated significantly with number of TFH markers positive (Spearman coefficient ρ=0.759). In summary, the stain panel chosen will have an impact on cases classified as PTCL-TFH. This entity may exist along a spectrum between PTCL-NOS and AITL.