Citations

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 Background: Inhibitors of HMG-CoA reductase (“statins”) are effective in reducing the risk of cardiovascular disease. Preclinical data from rodents suggested that they may increase the incidence of some cancers, including lymphoma. However, monitoring of randomized trials in humans have generally found decreased cancer risks, and these findings have been supported by a growing number of observational studies, including three recent reports of a protective association in NHL. The suggested anti-carcinogenic mechanisms for statins include their impact on inflammatory (including a shift from a Th1 to Th2 profile) and angiogenesis pathways, as well as the induction of apoptosis by modulation of signaling pathways, all of which are of biologic relevance in NHL. Methods: We evaluated the history of statin use and risk of NHL in a clinic-based study of 243 newly diagnosed NHL cases and 499 frequency matched controls enrolled at the Mayo Clinic from 2002-2005. Risk factor data were collected using self-administered questionnaires, and included history of use of cholesterol lowering drugs two or more years before diagnosis (for cases) or enrollment (for controls); specific drug names were not available. Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI), adjusting for age, gender, and residence. NHL subtypes were centrally reviewed, and subtype-specific risks were estimated using polychotomous logistic regression. Results. The mean age at diagnosis was 59.5 years for cases and 55% were men; among controls, the mean age at enrollment was 61.8 years and 54% were men. Twenty-one percent of the cases reported ever using statins compared to 30% of the controls, supporting a lower risk of NHL for ever users (OR=0.69; 95% CI 0.47–1.00). The lowest risk was seen for the longest users; i.e., compared to never users, person who had 10 or more years of use had the greatest reduction in risk (OR=0.52; 95% CI 0.24–1.10). A history of high cholesterol was also inversely associated with risk of NHL (OR=0.72; 95% CI 0.55–0.93). However, the association for the type of treatment for cholesterol lowering varied. Those who were medically treated had inverse association with risk of NHL (OR=0.63; 95% CI 0.46–0.86), while the no treatment and dietary change only groups were found to have no association with risk. The reduction in NHL risk with medical treatment was specific to statin use, as the use of other cholesterol lowering drugs was not associated with risk (OR=1.13; 95% CI 0.50–2.57). These results were not confounded by education, family history of NHL, body mass index, cigarette smoking or alcohol use. In subtype analysis, the inverse association was seen for diffuse large B-cell lymphoma (OR=0.27; 95% CI 0.08–0.90), but not for CLL/SLL or follicular NHL. Conclusions. Statin use was associated with a lower risk of developing NHL, particular diffuse large B-Cell lymphoma.

Abstract MALT lymphoma is a genetically unique disorder and five mutually exclusive chromosomal translocations have been identified thus far in this disease: t(11;18), t(14;18), t(1;14), t(1;2) and t(3;14). Despite this genetic heterogeneity, all but one of the translocations affect the NF-κB signaling pathway, which is critical if not essential for antigen receptor mediated B- and T-cell activation and likely enhances MALT lymphoma growth. However, the known translocations are present in only 25% of cases suggesting that additional uncharacterized translocations exist. We used long-distance inverse polymerase chain reaction (LDI-PCR) technique to clone a novel IGH translocation partner in a 60-year-old female with a history of Sjogren’s syndrome and primary MALT lymphoma involving the parotid gland. The breakpoint on chromosome 14 occurred within the IGHSA2 switch region while the breakpoint on chromosome X fell within Xp11.4. The breakpoint on chromosome Xp11.2 fell between two genes, GPR82 and GPR34, both of which code for orphan G-protein coupled receptors (GPCRs). The breakpoint also fell within and disrupted a larger gene called CASK (a membrane-associated guanylate kinase-MAGUK). To determine the prevalence of this translocation in MALT lymphoma we performed interphase FISH studies on 64 MALT lymphomas using a breakapart probe for GPR82. Only the index case had an abnormal split signal pattern. We next designed primers to perform real-time quantitative RT-PCR for the genes located on Xp11 and found that GPR34 RNA was highly expressed, a 49-fold increase, in the patient carrying the t(X;14)(p11.4;q32) translocation compared to a normal splenic B cell control. Expression of GPR82 and CASK RNA was similar between normal B cells and the patient carrying the t(X;14)(p11.4;q32) translocation. These data suggest that the GPR34 gene is dysregulated upon its translocation to the IGHSA2 switch region. We then measured GPR34 RNA expression in a panel of MALT lymphomas (n=12) and found that GPR34 was expressed at levels higher than that seen in normal B cells with an average increase of 11 fold, 9/11 of which had an expression lever greater than 3-fold over normal splenic B cells. In a gastric MALT lymphoma specimen arising in a 67-year-old male, we saw a 64 fold increase in GPR34 expression. Interphase FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. GPR34 RNA was also detected in other normal B cell populations and histologic subtypes of NHL but not to the extent seen in MALT lymphoma; values represent the expression of GPR34 normalized to β-actin and a normal B cell control (value of 1.0): t(X;14)(p11.4;q32) specimen, 49.0; MALT lymphoma, 11.0 (n=12); peripheral blood B cells, 0.48 (n=2); normal bone marrow B cells, 0.97 (n=3); follicular lymphoma, 2.47 (n=3); marginal zone B cell lymphoma, 2.47 (n=3); diffuse large cell lymphoma, 0.36, (n=3); mantle cell lymphoma, 2.67 (n=3); and multiple myeloma 0.52 (n=6). The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are particularly abundant in mast cells while lower levels were detected in other immune cells including B cells. However, little is known about its natural ligand, biologic function, or the signaling cascades activated by its engagement. Because the NF-κB signaling pathway has been shown to be a common downstream target of MALT lymphoma translocations we first examined the impact of GPR34 expression on phosphorylation of Iκ-Bα. Transient expression of a YFP-GPR34 expression plasmid in HeLa cells results in increased phosphorylation of Iκ-Bα compared to the YFP control. Additionally, we observed increased phosphorylation of ERK1/2 in GPR34-expressing cells, however no change in phosphorylation of GSK3β was detected. In summary, these data identify a novel IGHS translocation partner in MALT lymphoma and suggest that dysregulation of GPR34 is commonly found in MALT lymphoma. Overexpression of GPR34 results in activation of the NF-κB and MAP kinase pathways and may be a novel mechanism by which MALT lymphoma occurs.

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 use of high-resolution aCGH allows for whole genome screening for copy number changes even in tumors that rarely produce metaphase spreads. The aim of this study was to characterize the aCGH findings in B-CLL, lung MALT lymphoma, Waldenström’s Macroglobulinemia (WM), Multiple Myeloma (MM) and human myeloma cell lines (HMCLs). This study includes 301 B-cell malignancy samples (19 B-CLL, 19 MALT, 20 WM, 48 HMCLs and 195 MM) and an additional 76 melanoma samples. We found MM to have the highest number of abnormalities per karyotype, with 15.8±7.5 (mean ± SD) and 20.6±17.0 in hyperdiploid (H) and non-hyperdiploid MM (NH), respectively. The remaining diseases have a significantly lower number of abnormalities: 5.5±4.5 in B-CLL, 3.5±1.96 in MALT and 4±3.5 in WM. Aneuploidy and biallelic deletions were rare events in these three diseases. B-CLL has two highly recurrent abnormalities involving 11q22-q23 (29%) and 13q14.3 (33%). A minimal deleted region (MDR) of ∼108Kb was identified at 11q comprising ACAT1, NPAT and ATM. In 13q, a 280Kb MDR was recognized, including TRIM13, KCNRG, mir-15a and mir-16. The subgroup of MALT patients with t(11;18) had very few abnormalities and the majority are a consequence of unbalanced translocations (MALT, BIRC2 and BIRC3 deletions). In the remaining MALT samples, trisomy 3 and 18q gain were the most common abnormalities (31% each). The 6q16.3-q26 deletion was the most common abnormality in WM (45%). In 4 of 9 patients with 6q deletion there was an associated 6p gain. The same observation was found in 2 of 3 patients with 8p loss and concomitant 8q gain. Interestingly, 6p and 8q gain were not identified in patients without 6q and 8p deletions. Deletion of 17p was the most common recurrent abnormality observed in our panel of B-cell cancers, being identified in MM, WM and B-CLL. In B-CLL and WM, small mono and biallelic deletions of the NF-kB negative regulators, TRAF3 and NFKBIA, were identified, suggesting a potential involvement of this pathway in the pathogenesis of these diseases. Hierarchical clustering of the aCGH data in MM identified 3 distinct groups, NH-MM, H-MM and MM with deletions of chromosome 13 and 14. Interestingly, MGUS and SMM were more commonly clustered with H-MM. Several sub-clusters were observed in the H-MM group, the most prominent being made up exclusively of patients from TC class D1. This cluster was characterized by the presence of trisomy 11 and 21. The 11q13 TC group clustered and was highly associated with 11q amplifications due to unbalanced t(11;14). The 6q deletion defined a cluster with WM, MM and Melanoma patient with that abnormality. In the case of B-CLL and MALT, the only identifiable clusters were observed in cases with 13q loss and trisomy 3, respectively. These results highlight both, the common and unique aCGH findings in several B-cell cancers. By hierarchical clustering, MM was significantly divergent from the rest of the B-cell diseases. Due to the limited number of abnormalities in B-CLL/MALT/WM, the clustering of these groups was exclusively based on the common abnormalities. The use of aCGH has refined the MDRs of 11q and 13q in B-CLL. Additionally, the identification of deletions affecting the NF-kB pathway in B-CLL and WM has prompted us to hypothesize on the importance of constitutive NF-κB activation in these diseases.

Abstract Purpose: Despite therapeutic advancements, biological markers that predict the natural history of primary central nervous system lymphoma (PCNSL) are lacking and age and performance status are the only consistently identified independent prognostic variables. BCL6 rearrangements and deletion of the tumor suppressor gene R-PTP-κ (PTPRK) at 6q22 are thought to be common genetic abnormalities in PCNSL but their prognostic significance is unknown. The aim of this study is to determine the prevalence and survival impact of del(6)(q22), BCL6, immunoglobulin heavy chain (IGH), and MYC gene rearrangements and prevalence of Epstein-Barr virus (EBV) infection in PCNSL affecting immunocompetent patients. Patients and methods: Seventy-six specimens from 76 HIV-negative, immunocompetent patients with PCNSL newly diagnosed and treated at Mayo Clinic between 1985 and 2006 were studied. Interphase fluorescence in-situ hybridization (FISH) was performed using two-color break apart probes (BAP) for BCL6 and MYC, a two-color dual-fusion probe for IGH-BCL6, and a two-color probe for del(6)(q22) on thin sections of paraffin-embedded tumor samples. Two-color IGH BAP FISH probes were also used to confirm IGH rearrangements in cases showing extra IGH signals without fusion using the IGH-BCL6 probe. In situ hybridization was performed using probes that recognize EBV-encoded RNA (EBER) on paraffin-embedded tumor samples. Survival data were analyzed for patients diagnosed after 1997 (n=53), corresponding to the change to high dose methotrexate as the standard of care. Survival was calculated from the date of tissue diagnosis to date of death or last contact. Survival curves were estimated using the Kaplan-Meier method. The log-rank test was used to compare survival across groups. Two-tailed p-values <0.05 were considered statistically significant. Clinical information for the 1985–2006 and 1997–2006 groups was comparable, including median age (67 years (y) vs 68 y), age range (26–87 y for both groups), percentage of deaths (67 vs 64) and median follow up for survivors (588 d vs 395 d). Results: Thirty-four (45%) cases showed del(6)(q22), 6 of which alsocontained a BCL6 rearrangement. Seventeen (22%) cases had a BCL6 rearrangement. Translocations involving IGH and an unknown partner gene (n=2) and MYC and an unknown partner gene (n=2) were also identified. All cases were EBER negative. Of the 53 patients diagnosed after 1997, 23 lacked del(6)(q22) or BCL6 rearrangement and had a median overall survival (MS) of 731 days (d). The 17 patients with an isolated del(6)(q22) had a MS of 90 d and the 13 patients with a BCL6 rearrangement had a MS of 442 d (p=0.0016). Conclusions: Del(6)(q22) and BCL6 rearrangements are common in PCNSL (45% and 22% of cases, respectively) and are associated with decreased survival, particularly del(6)(q22) seemingly independent of patient age and treatment time trends. IGH translocations were less frequent than in systemic diffuse large B-cell lymphoma (13% vs 51%), suggesting a distinct pathogenesis. MYC translocations and EBV infection are rare in PCNSL affecting immunocompetent patients.

PCNSL molecular pathogenesis in immunocompetent patients is largely unknown. Small studies suggest a role for immunoglobulin heavy chain (IGH) gene translocations. We studied the incidence of IGH, BCL6, and MYC gene rearrangements in 44 PCNSL affecting immunocompetent patients, diagnosed and treated at Mayo Clinic (1992–2006). All cases were DLBL according to the WHO classification, confined to the CNS. Interphase fluorescence in‐situ hybridization (FISH) was performed using a two‐color IGH‐BCL6 dual‐fusion probe and a two‐color MYC breakapart (BAP) probe on paraffin‐embedded tumor samples. Two‐color BCL6 or IGH BAP FISH probes were also used in cases showing extra FISH signals other than IGH‐BCL6 fusion. Sufficient tumor cells for FISH analysis using the IGH‐BCL6 and MYC probes were available in 43 and 39 cases, respectively. IGH‐BCL6 fusion was present in 6 (14%) cases, one also with a separated MYC signal. Three (7%) cases showed 3 intact BCL6 signals using both the IGH‐BCL6 and BCL6 BAP probes, indicating trisomy 3. One (2%) showed 3 intact IGH signals using both the IGH‐BCL6 and IGH BAP probes, indicating trisomy 14. Remaining 33 (77%) cases lacked abnormalities involving IGH, BCL6 or MYC. IGH‐BCL6 fusion was present in 14% of cases, 1 of which also had a translocation involving MYC and an unknown partner gene. All other cases lacked translocations involving IGH, BCL6, and MYC. The frequency of IGH‐BCL6 translocations in PCNSL is less than that seen in systemic DLBCL. Other translocations involving IGH that are not uncommon in systemic DLBCL, such as IGH‐BCL2 and IGH‐MYC, are rare to absent in PCNSL, suggesting a distinct pathogenesis for PCNSL compared to systemic DLBCL.

AbstractBackgroundConventional primary pelvic chondrosarcoma often presents as a low‐ or intermediate‐grade tumor in older patients. Although this is the most common variant of pelvic chondrosarcoma, studies examining treatment outcomes are lacking. The purpose of this study was to evaluate patients with these tumors to determine their outcomes of treatment.MethodsSeventy‐three patients (grade I [n = 19, 26%] and grade II [n = 54, 74%]) were reviewed including 55 (75%) males and 18 (25%) females, with a mean age of 51 (range, 17‐81) years and follow‐up of 9 ± 5 years.ResultsThe 10‐year disease‐specific survival was 71%. Grade II disease (hazard ratio [HR], 6.74; P = .04) and age ≥50 years (HR, 3.97; P = .02) was associated with death due to disease. The 10‐year local recurrence‐ and metastatic‐free survival were 79% and 72%. Of the patients with a local recurrence (n = 11), 7 (64%) recurred at a higher histological grade. Patient age ≥50 years was associated with local recurrence (HR, 10.03; P = .02) and metastatic disease (HR, 4.20; P = .02).ConclusionAdvancing patient age was an independent risk factor for worse survival and disease recurrence. Tumors often recurred locally at a higher grade and as such wide local excision remains the treatment of choice for these tumors.