Immunoglobulin Lambda Light Chain Research Articles

Abstract 1989: Gene expression signature of the non-cancerous liver tissue associated with the early recurrence of hepatocellular carcinoma

Abstract Recurrence of hepatocellular carcinoma (HCC) might be associated with biological aggressiveness of the cancer as well as background status of the liver. In this study, the predictive values for the early recurrence were assessed on patients with HCC after curative hepatectomy using clinicopathological factors and genome-wide gene expression signatures. Multivariate analysis of clinico-pathological factors revealed portal vein invasion (p=0.007), hepatitis C virus infection (p=0.001), and low levels of serum albumin (p=0.003) as the risk factors, yielding a ROC-plot AUC value of 0.854 with 61.5% sensitivity and 81.8% specificity. Analysis of the gene expression profiling on the cancer tissues of HCC revealed the significant differences in CD24 stem cell marker and MTSS1 metastasis suppressor molecule. The cancer six-gene signature for the prediction of recurrence yielded a ROC-plot AUC value of 0.897 with 81.8% sensitivity and 88.5% specificity. Using the non-cancerous liver tissues surrounding HCC, distinct differences were recognized by the gene expression profiling such as IGL immunoglobulin lambda light chain and MAP2K1 mitogen-activated protein kinase kinase. The non-cancer six-gene signature yielded a ROC-plot AUC value of 0.966 with 84.8% sensitivity and 88.5% specificity for the prediction of HCC recurrence. These gene expression signatures can be involved in the metastatic potentials of the invasive HCC, and the multi-centric hepatocarcinogenesis, respectively. Our results might indicate the molecular targets for prediction and/or treatment of the recurrence, as well as the super-high risk group of HCC within the patients with hepatitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1989.

Analysis of DDT Isomers with Enzyme-Linked Immunosorbent Assay and Optical Immunosensor Based on Rat Monoclonal Antibodies as Biological Recognition Elements

New rat monoclonal antibodies (mAbs) for DDT [1,1,1 -trichloro-2,2-bis (4-chlorophenyl) ethane], namely DDT 7C12, DDT 1C1, and DDT 1B2, were developed, characterized, and applied in ELISA both in coating antigen and in enzyme-tracer format. The latter used horseradish peroxidase (HRP) or glucose oxidase as enzymes. The lowest concentration of p,p'-DDT was determined with mAb DDT 7C12 and DDT-hapten HRP, with a test midpoint (IC50) of 0.5 +/- 0.2 microg/L (n=10) in 40 mM PBS (phosphate-buffered saline). The mouse anti-rat immunoglobulin lambda-light chain mAb LA1B12 was used as capture mAb. The best IC50 for o,p'-DDT in 40 mM PBS was 1.0 +/- 0.3 microg/L (n=12) and was obtained with mAb DDT 1C1 and DDT-hapten HRP, whereas mAb DDT 1B2 was very selective for p,p'-DDT with an IC50 of 4.2 + 1.6 microg/L (in 40 mM PBS, n=9). An optical immunosensor was optimized and applied for the analysis of DDT (or DDT equivalents). This immunosensor consists of a bench-top optical readout device and disposable sensor chips, which include the fluidic system. Evanescent field excitation and emission of the fluorophore Oyster-645 was used. An IC50 for p,p'-DDT [in 5% (v/v) isopropanol in 40 mM PBS] of 4 microg/L was obtained using DDT 7C12-Oyster-645. ELISA and immunosensor were used for the analysis of p,p'-DDT in unspiked and spiked surface water samples. Within the working ranges of these immunotechniques, recoveries ranged from 80 to 120%.

Detail Characterization of 22q11 Deletion and Mir-650 Role in CLL Patients.

Abstract 1231Poster Board I-253 BackgroundChromosomal abnormalities are present in the majority of CLL cases (>80%) and recently, a novel deletion of 22q11 was described in CLL patients. This locus encodes lambda immunoglobulin light chain (IgL) subgenes and several protein-coding genes, which distinguishes IgL from kappa Ig or heavy Ig locus. Gunn et al. (2008) previously characterized the minimally deleted region of 22q11 in CLL as 0,34Mb (containing PRAME, ZNF280A, ZNF280B, GGTLC2) and described the PRAME as a candidate gene with prognostic impact in CLL. Interestingly, the IgL variable segment (V2-8) located at 22q11 includes an annotated miR gene miR-650. According to the performed BLAST search also other subgenes of V2 family include homologues for miR-650, which overlay the leader exon of the V2 subgenes. This could be of particular importance because the expression of miRNAs was previously shown to be associated with known prognostic markers in CLL: 13q deletion, IgVH status (Calin, 2002, 2005) and p53 deletion/mutation (Mraz, 2009). Moreover, miRNA pathways are involved in the regulation of immune system functions including IgG production and V(D)J recombination (Vigorito, 2007; Koralov, 2008). AimWe analyzed genomes of 40 CLL patients using array-CGH (CGH Microarray 4×44 K, Agilent) and FISH (del13q14, del17p13, del11q23, +12), and in particular focused on detailed characterization of 22q11 deletion. An expression analyses (RT-qPCR, TaqMan miRNA Assay, ABI) was performed for protein-coding genes and miR-650 located at 22q11. ResultsUsing array-CGH additional aberrations to FISH were found in the majority of samples and among them, the deletion of 22q11 locus was observed most often (ranging in size up to ∼ 0,77 Mb). Altogether, 40% of patients (16/40) had detectable deletion in this locus and according to our finding, all observed deletions were a consequence of a VJ rearrangement event, which lead to an elimination of specific genes located between VJ elements. These rearrangements were characterized in detail by PCR detection (BIOMED-2 protocol) and sequencing. In our cohort, deletions of 22q11 containing PRAME gene constituted only ∼44% (n=7) and 56% patients (n=9) harbored smaller deletions not involving PRAME. To analyze the gene expression, we performed a Real-Time PCR analyses for previously mentioned protein-coding genes and miR-650. The Real-Time PCR assay for miR-650 reported higher expression of miR-650 in CLL cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=5) compared to samples utilizing different V lambda family or expressing kappa Ig (n=36). It remains to be elucidated how and whether all the V2 subgenes give rise to a mature and functional miRNA. The overlap between miR-650 and the leader exon of IgL gene could provide a unique mechanism for coordinated IgL and miRNA expression. To assess the function of miR-650 we performed a microarray (Human OneArray, Phalanx) expression profiling 24/48 hours post transfection of CLL cells with a short RNA mimicing miR-650 (miRIDIAN Mimic, Dharmacon). The transfection led to a significant down-regulation of dozens of mRNAs that could be considered as potential targets for miR-650. Interestingly, the predicted targets for miR-650 include early B cell factor (EBF3; target with highest total score – TargetScan) and VPREB1 (pre B lymphocyte gene 1) whose expression is important for immature phenotype of pre-B lymphocytes prior to light chain recombination. The coupled activation of miR-650 and lambda Ig expression following productive rearrangement of IgL locus could potentially affect the expression of VPREB1/EBF3. ConclusionDeletion of 22q11 is frequently detected in CLL patients and we suggest that it is caused by physiological process of lambda immunoglobulin variable locus rearrangement. This leads to the elimination of protein-coding genes located between VJ elements. Interestingly, the V2 family of variable segments for lambda light chain at 22q11 includes an annotated miR gene miR-650 and its homologues. The productive lambda locus rearrangement utilizing V2 family could be also coupled with activation of miR-650 expression with consequent relevance for B cell biology. This work is in progress and in further analyses we expect complex characterization of miR-650 functions in B cells and CLL. Supported by IGA MZ CR NR9293-3/2007 and IGA MZ CR NS10439-3/2009. DisclosuresMayer:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.

Elevated Pre-Treatment Serum Immunoglobulin Free Light Chains (FLC) Are Associated with Poor Event-Free and Overall Survival in Diffuse Large B-Cell Lymphoma (DLBCL).

Abstract 136 Background:The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. This assay has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and AL amyloidoisis, and has been incorporated in response criteria for both MM and AL amyloidosis. A percentage of non-Hodgkin's lymphoma (NHL) patients also present with abnormal FLC concentrations and recent data suggest that AIDS patients with elevated serum FLC are at higher risk of developing NHL. Here we assess the impact of FLC in a cohort of patients with diffuse large B-cell lymphoma (DLBCL) enrolled on a cooperative group clinical trial. Methods:Newly diagnosed DLBCL patients were treated with epratuzumab + R-CHOP (ER-CHOP) on NCCTG clinical trial N0489. Serum FLC was quantitated prior to treatment and after cycles 2 and 6 using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Patients were followed per clinical trial protocol for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Results:107 patients were enrolled on the trial. 80 patients had a pre-treatment serum draw and a central pathology confirmed diagnosis of DLBCL, of which 47 had a post-cycle 2 sample (C2) and a 54 had a post-cycle 6 sample (C6). At a medium follow-up of 25 months (range 7-39), 22 patients (28%) had an event and 17 patients died (21%). Prior to treatment 23 patients (29%) had abnormal kappa concentrations (all elevated), 16 (20%) had abnormal lambda concentrations (11 elevated, 5 decreased), and 9 (11%) had an abnormal kappa/lambda ratio. FLC concentrations were significantly reduced in patients during treatment, with a median reduction in kappa (C2: 46%, C6: 64%) and lambda (C2: 44%, C6: 49%), all p<0.0001. Elevated FLC prior to treatment was highly associated with both OS (logrank p=0.009) and EFS (logrank p=0.006); these associations remained significant after adjusting for IPI (both p = 0.01). Conclusions:A significant percentage of DLBCL patients present with abnormal FLC concentrations. Kappa and lambda are significantly reduced for most patients during treatment. Elevated pre-treatment FLC concentrations are associated with inferior EFS and OS in DLBCL and this association is independent of the IPI. A follow-up study of FLC and prognosis is currently underway in a cohort of 1500 NHL patients from the Mayo Clinic and University of Iowa SPORE.Association of Pre-treatment FLC with OutcomeN (%)OS HR (95% CI)EFS HR (95% CI)Elevated KappaUnivariate23 (29%)3.3 (1.3, 8.5)2.5 (1.1, 5.9)IPI Adjusted3.1 (1.2, 8.1)2.3 (1.0, 5.5)Elevated LambdaUnivariate11 (14%)3.1 (1.1, 8.8)4.2 (1.7, 10.4)IPI Adjusted4.4 (1.5, 13.2)5.3 (2.1, 13.5)Abnormal Kappa/Lambda RatioUnivariate9 (11%)2.8 (0.91, 8.6)1.9 (0.65, 5.7)IPI Adjusted4.3 (1.4, 14.0)2.5 (0.82, 7.8)Elevated FLC (either)Univariate26 (33%)3.4 (1.3, 8.9)3.1 (1.3, 7.2)IPI Adjusted3.3 (1.3, 8.8)2.9 (1.3, 6.8) [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.

A Case of Lambda-expressing Pulmonary MALT Lymphoma with Dual Clonal Rearrangements of Kappa and Lambda Immunoglobulin Light Chain Gene

A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin‐6 to induce the expression of recombination‐activating gene in B lymphocytes

B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin kappa and lambda light chain expression and by ligation-mediated-PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the kappa-to-lambda ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA.

Primary Antral Duodenal Extramedullary Plasmacytoma Presenting With Melena

A 69-year old African woman presented with a 2-week history of fatigue and melena. Her medical history was notable for congestive heart failure complicated by an apical thrombus requiring Coumadin. Physical examination showed pallor, but was otherwise unremarkable. Abnormal laboratory values included a hemoglobin level of 4.4 g/dL and an international normalized ratio of 2.3. An upper gastrointestinal endoscopy showed an erosive antral gastropathy (Figure A) and an ulcerated mass located on the posterior-superior wall of the duodenum (Figure B). Biopsy specimens from the antrum and duodenum showed a plasma cell infiltrate with plasmablastic features (Figure C, 400×, H&E). Neoplastic cells were reactive for the plasma cell marker CD138 and found by in situ hybridization to be lambda immunoglobulin light chain restricted (Figure C). CD20, a B-cell marker, was not expressed by the malignant cells. The morphology in conjunction with the immunohistochemistry was most consistent with an extramedullary plasmacytoma, and the negativity for the B-cell marker CD20 excluded the possibility of marginal zone B-cell lymphoma of mucosa associated lymphoid tissue lymphoma. A subsequent bone marrow biopsy showed a normo-cellular marrow with trilineage hematopoiesis with less than 5% plasma cells. Serum electrophoresis and immunofixation showed an M-spike of immunoglobulin A lambda, a finding identified in approximately 25% of plasmacytoma cases.1 Together, these findings confirmed a final diagnosis of a primary extramedullary plasmacytoma of the duodenum and stomach as the cause of her melena. Coumadin therapy was stopped, which limited her bleeding. Due to cardiac and renal comorbidities, the patient was deemed a poor candidate for surgical intervention or chemotherapy. Extra-corporeal radiation therapy was begun and initially reduced the overall size of her duodenal plasmacytoma. However, because of its location it ultimately progressed to a state of biliary obstruction. At endoscopic retrograde cholangiopancreatography, the bile duct was inaccessible secondary to tumor burden. The patient required a percutaneous transhepatic biliary drainage. Extramedullary plasmacytoma is a rare entity accounting for less than 4% of all plasma cell tumors and occurs mainly in the upper aerodigestive tract. In the gastrointestinal tract, most tumors occur in the stomach, whereas only 20% to 30% arise in the small intestine.2 No general consensus guidelines for treatment currently exist. However, based on the well-known radiation sensitivity of plasma cell tumors, radiotherapy is accepted as a standard therapy.3 Surgery for extramedullary plasmacytomas of the small intestine can also offer cure and may be the preferred treatment for some patients.4 If complete surgical resection is not feasible, then combined radiation or chemotherapy is recommended. The overall disease prognosis is good, with a 10-year overall survival of 70%.5 In a review of 721 cases by Alexiou et al,3 after treatment, approximately 65% of patients were free of recurrence and did not progress to multiple myeloma, 21% experienced recurrence, and 14% of cases evolved to multiple myeloma.

Novel Isolated Cerebral ALλ Amyloid Angiopathy With Widespread Subcortical Distribution and Leukoencephalopathy Due to Atypical Monoclonal Plasma Cell Proliferation, and Terminal Systemic Gammopathy

Neuropathologic and biochemical findings in a 34-year-old man whose disease began 2 years before death appearing as chronic progressive encephalitis and culminated in mutism are reported. Cerebrospinal fluid and serum of the patient showed a brain-restricted monoclonal lambda-light chain apparently produced by a small monoclonal immunoglobulin Glambda plasma cell population. In the preterminal stage, there was a systemic monoclonal gammopathy, the source of which could not be identified. At autopsy, there was extensive amyloid deposition in most vessels throughout the cerebral and cerebellar white matter, basal ganglia, and thalamus and diffuse leukoencephalopathy; cerebral and cerebellar cortices, other portions of the CNS, and non-CNS tissues were spared. Partial amino acid sequence analysis demonstrated that the amyloidogenic protein originated from immunoglobulin lambda-light chains which were produced by monoclonal plasma cells. There are 2 similar cases reported in the literature. The distribution of ALlambda deposits in these 3 cases indicates that widespread subcortical vascular amyloidosis with leukoencephalopathy is a novel clinicopathologic entity distinguished from other cerebral diseases with local amyloid light chain deposition, including amyloidoma, leptomeningeal vascular amyloidosis, solitary intracerebral plasmacytoma, primary intracerebral lymphoma with plasmacytic differentiation, and multiple sclerosis with demyelination-associated amyloid deposition.

Serum free light chains

Background Serum free light chains (FLC) assay measures the concentration of free kappa and lambda immunoglobulin light chains. Clinical uses FLC assay is clinically indicated for screening and prognosis of multiple myeloma and related plasma cell disorders including oligosecretory disease, AL amyloidosis and monoclonal gammopathy of undetermined significance. There are no data to support its use in myeloma with measurable disease by other methods. In assessment of disease response to treatment, FLC measurement is recommended in documenting stringent complete response in multiple myeloma, and in oligosecretory diseases but is unvalidated in light chain myeloma and intact immunoglobulin disease. Analytical issues Use of polyclonal FLC antibodies raises the question of adequate specificity and binding affinity to measure monoclonal FLC. Problematic samples can give FLC under- or over-estimation due to sample dilution anomalies, giving rise to difficulties in result interpretation, particularly when monitoring patients with clonal plasma cell diseases. Laboratory staff and clinicians should be aware of the potential for non-reactivity of individual monoclonal FLC, the effect that dilution has on FLC measurement, and the impact of assay imprecision on result interpretation. These issues, if not adequately appreciated, have the potential to mislead clinical diagnosis and assessment of response to therapy.

Spontaneous rupture of the liver in a patient with systemic AL amyloidosis undergoing treatment with high-dose melphalan and autologous stem cell transplantation: A case report with literature review

A 55-year-old woman with primary Immunoglobulin light chain (AL) systemic amyloidosis died due to spontaneous rupture of her liver following treatment with high-dose melphalan and autologous stem cell transplant (HDM/SCT). She was first diagnosed after developing nephrotic-range proteinuria. Spontaneous rupture of her liver occurred 10 days after treatment with HDM/SCT and was complicated by septic shock. She was not eligible for surgical intervention and died shortly after. Amyloid fibrils were extracted from the autopsied liver sample (05-135L) and the biochemical nature of the fibrils was analyzed using electrophoretic and immunohistochemical techniques. Our testing showed that the fibrils were composed of immunoglobulin lambda light chains that were not glycosylated.While the liver is often involved in AL amyloidosis, this is the first documented case of a spontaneous hepatic rupture in a patient during treatment with HDM/SCT. A literature review of spontaneous liver rupture in patients with amyloidosis is presented.

Proteomic analysis of cerebrospinal fluid from patients with idiopathic temporal lobe epilepsy

Proteomic analysis of cerebrospinal fluid (CSF) from patients with temporal lobe epilepsy (TLE) and controls was carried out using two-dimensional gel electrophoresis followed by liquid chromatography electrospray ionization tandem mass spectrometry. Five protein spots showed significant differential expression ( p < 0.05): vitamin D-binding protein (DBP) was elevated in the CSF of TLE patients whereas cathepsin D, apolipoprotein J, Fam3c, and superoxide dismutase 1 (SOD1) were decreased in the CSF of TLE patients. Additional six protein spots presented only in the CSF of epilepsy patients were identified as tetranectin (TN), talin-2, apolipoprotein E, immunoglobulin lambda light chain (IGL@), immunoglobulin kappa variable light chain 1–5 (IGKV1–5), and procollagen C-endopeptidase enhancer 1 (PCOLCE). Expression of DBP, SOD1 and talin-2 was validated by western blot. Our results may provide better understanding of the pathophysiologic mechanisms underlying epileptogenesis and possible epilepsy biomarkers.

Glomeruloid haemangioma is not always associated with POEMS syndrome

Glomeruloid haemangioma is considered a specific marker of POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes) syndrome and it is usually but not always associated with multicentric Castleman's disease. We report a 78-year-old man who presented with a single, red-violet soft nodule with superficial telangiectases on the scalp. Histopathologically, the lesion consisted of lobules of coiled aggregated capillaries that involved the lumina of dilated vascular structures, mimicking renal glomeruli. A collagenous stroma separated the capillary lobules, and eosinophilic, periodic-acid-Schiff positive globules of varying sizes and shapes were seen within the cytoplasm of endothelial cells. Immunohistochemical studies with antibodies against IgA and IgG, and against the kappa and lambda light chains of immunoglobulins showed immunoreactivity within the eosinophilic globules. Results of complete blood count, liver, renal and thyroid function tests, fasting blood sugar measurement, serum levels of oestradiol, testosterone, prolactin and cortisol, serum protein electrophoresis, immunoelectrophoresis and immunofixation yielded normal or negative results. No Bence-Jones proteinuria was found in a sample from a 24-h urine collection. To our knowledge, only two cases of glomeruloid haemangioma have been previously reported in patients without POEMS syndrome. We describe the third case of glomeruloid haemangioma in a patient without features of POEMS syndrome.

Expression of Myeloma-Specific Markers in Bone Marrow Spicules Using a Novel Immunohistochemical Technique

In addition to its role in diagnosis and assessing the prognosis of numerous malignancies, immunohistochemistry (IHC) is an important tool for the analysis of protein biomarker expression in tissue sections. The combination of bone marrow (BM) biopsy and IHC is frequently used to study histopathological alterations in diseases such as multiple myeloma (MM). However, due to the harsh decalcification process generally used for processing of bone marrow biopsies, protein epitopes are occasionally rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin-clot matrix that allows for IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Briefly, bone marrow aspirates drawn from MM patients were subjected to a brief processing step to isolate the marrow spicules. Once isolated, the spicules were admixed with thromboplastin and pooled normal human plasma to create a clot. The clot was allowed to harden at 37°C after which it was placed into an embedding cassette for fixation and histological processing. The result was a formalin-fixed, paraffin-embedded clot suitable for IHC studies, including the construction of TMAs. Using patient paired spicule-clot and core biopsies from patients diagnosed with myeloma, we studied several antibodies including, but not limited to, kappa and lambda immunoglobulin light chains, CD138 and Cyr61, a member of the CCN family of extracellular matrix-associated signaling proteins. Results demonstrated that IHC staining for all antibodies was comparable in both the spicules and in the cores. While BM core biopsies had areas of non specific staining for several antibodies, this was not generally observed in the BM spicule samples with the exception of immunoglobulin light chains which displayed an element of non-specific staining with both samples. Moreover, we observed a consistently superior morphology in the BM spicule samples than in matched bone marrow core biopsies. In conclusion, our study demonstrates that BM spicule samples processed from MM patients using this novel fibrin-clot matrix technique were suitable for IHC and had generally lower background and non-specific staining coupled with better morphology when compared to matched core biopsies.

Array CGH Analysis of Chronic Lymphocytic Leukemia Reveals Frequent Cryptic Monoallelic and Biallelic Deletions of Chromosome 22q11.

Bacterial artificial chromosome array-based comparative genomic hybridization (array CGH) was used to detect recurrent genomic imbalances in 187 CLL cases. Detection rates of clinically relevant chromosomal abnormalities involving losses of 11q23, 13q14, 17p13, and whole chromosome 12 gains were consistent with previously reported results. However, submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most commonly detected recurrent chromosomal aberration in CLL. The del(22)(q11) occurred as the sole genomic alteration in three cases, but was associated with additional copy number changes in 25 cases, most commonly with the 13q14 deletion, where it was found in 17 of the 28 cases (61%). Twenty-four cases exhibited monoallelic 22q11 losses, four cases showed biallelic losses, and two cases also exhibited concomitant gains of whole chromosome 22.Breakpoint mapping using oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb, and all cases included a minimally deleted 340,279 bp region located within the lambda immunoglobulin light chain variable region locus. The minimally deleted region included four protein coding genes, including the closely related zinc finger protein genes ZNF280A and ZNF280B, the gamma-glutamyltransferase light chain 2 (GGTLC2) gene, and the preferentially expressed antigen in melanoma (PRAME) locus. Quantitative real-time PCR expression studies for these four genes revealed that the expression of ZNF280A, ZNF280B, and PRAME mRNA was significantly lower in the 22q11 deletion cases compared to non-deleted cases by both the two sample t-test and the Wilcoxon rank sum test methods. Mean expression of ZNF280A mRNA was 26-fold lower in the del(22) (q11) samples compared to non-deleted samples (t-test, p=0.02612; Wilcoxon rank sum test, p=0.002511). Mean expression of ZNF280B mRNA was decreased by 11-fold in the del(22)(q11) samples compared to non-deleted samples (t-test, p=0.01442; Wilcoxon rank sum test, p=9.768x10−6). Mean expression of PRAME mRNA was 11-fold lower in the del(22)(q11) cases compared to non-deleted cases (t-test, p=0.005517; Wilcoxon rank sum test, p=0.0317). However, mean expression of GGTLC2 mRNA was only 1.6-fold lower in the del(22)(q11) cases compared to non-deleted cases. This change was marginally significant by t-test (p=0.06718) and not significantly different by the Wilcoxon rank sum test (p=0.1329). CLL cases with a biallelic del(22)(q11) exhibited the lowest levels of expression of ZNF280A, ZNF280B, and PRAME mRNA.The ZNF280A and ZNF280B genes are closely related zinc finger proteins which exhibit significant sequence similarity to the Drosophila Suppressor of Hairy-wing (Su[Hw]) protein, a leucine zipper protein that contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators (independent transcriptional domains). To date, there is no direct evidence linking these two zinc finger proteins with human cancers. However, it is conceivable that alterations in the copy number of these genes could have a significant effect on gene regulation in CLL, and possibly other human cancers.The PRAME gene encodes a membrane- and cytoplasm-associated protein that was first described as a tumor antigen in melanoma that triggered autologous cytotoxic T-cellmediated immune responses. High levels of PRAME mRNA expression have been described in a number of human malignancies, including CLL. High PRAME expression is present in acute leukemias with a more favorable prognosis such as AML with the t(8;21), AML-M3 with the t(15;17), and childhood B-ALL with or without the t(12;21). These findings have made PRAME an attractive candidate for immunotherapy and a potentially useful marker for minimal residual disease monitoring. Our study is the first to correlate submicroscopic deletions of the PRAME locus with decreased expression of PRAME mRNA in CLL. The lack of reciprocal duplications and the presence of biallelic deletions also point to a possible role for loss of the PRAME gene locus in the pathogenesis of CLL.Further studies are needed to assess the prognostic significance of submicroscopic 22q11 deletions and to determine whether these alterations contribute to the pathogenesis of CLL.

Hyperplasia of Hair Follicles and Other Adnexal Structures in Cutaneous Lymphoproliferative Disorders

We studied 53 cutaneous lymphoproliferative disorders, all of which manifested hair follicle hyperplasia. There were 42 cases conforming to the description of pseudolymphomatous folliculitis (PLF) and 11 cases of authentic lymphomas including mycosis fungoides, CD30+ anaplastic large cell lymphoma, diffuse large B-cell lymphoma, B-cell small cell lymphoma/leukemia, and peripheral T-cell lymphoma, not otherwise specified. All patients with PLF clinically presented with a solitary nodule preferentially involving the face. Beside hair follicle hyperplasia, the typical features were a dense infiltrate of small well-differentiated lymphocytes, lymphoplasmacytoid cells, plasma cells, and epithelioid histiocytes forming tiny granulomas. Some unusual or worrisome features recognized included eccrine/apocrine duct hyperplasia, subcutis/muscle infiltration, lymphocyte "smudging," single file infiltration, and large atypical cells. Immunohistochemically, T-cell predominant cases dominated in the series. All 34 tested cases revealed a polyclonal pattern of kappa and lambda immunoglobulin (Ig) light chain expression. In 4 cases, scattered CD30+ cells were identified. Monoclonal rearrangements of T-cell receptor (TCR) and IgH genes were detected in 19 and 3 cases respectively, including 1 case with dual T-cell receptor/IgH rearrangement. Three of 30 tested cases proved positive for herpes simplex virus-1, whereas herpes simplex virus-2 always tested negative. Of 31 cases tested for Borrelia burgdorferi, 30 specimens were negative. In 9 cases, fluorescent in situ hybridization for t(11;18) and t(14;18) revealed none of the above translocations. The most common treatment modality was surgical removal. Forty patients with a mean follow-up of 3.7 years included 39 patients with no evidence of disease and 1 individual with local recurrence. The comparison of "clonal cases of PLF" and those with polyclonal population or in which clonality remained undetermined revealed no differences between the 2 groups in the clinical presentation, pathologic, and immunohistochemical features. We conclude that hyperplasia of hair follicles and other adnexa can be seen not only in the condition currently known as PLF, but also in genuine cutaneous lymphomas and may be just a happenstance secondary to a basic pathologic process.

Translocation (18;22)(q21;q11) in B-cell lymphomas: a report of 4 cases and review of the literature

Follicular lymphomas characteristically carry t(14;18)(q32;q21) which results in IGH-BCL-2 fusion. Variant translocations that juxtapose the BCL-2 gene with the immunoglobulin kappa (2p11) and lambda (22q11) light chain genes are rare. We report 4 cases of B-cell lymphoma/leukemia associated with t(18;22)(q21;q11). The t(18;22)(q21;q11) was the sole aberration identified by conventional cytogenetics in 2 cases. Three cases were classified as chronic lymphocytic leukemia, and one as follicular lymphoma based on morphology and immunophenotype. Fluorescence in situ hybridization analysis was performed on all 4 cases using a BCL-2 breakapart probe. The BCL-2 gene was rearranged in all cases. Immunoglobulin lambda light chain gene rearrangement was shown in 3 cases using bacterial artificial chromosome probes spanning the variable and constant clusters of the IGlambda gene. Each case was negative for MALT-1 rearrangement using a MALT-1 breakapart probe. These cases illustrate that t(18;22)(q21;q11) is more commonly observed in chronic lymphocytic leukemia and may represent either an initial or secondary genetic event.

Characterization of the bovine immunoglobulin lambda light chain constant IGLC genes

To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals.

Immunoglobulin free light chain ratio is an independent risk factor for progression of smoldering (asymptomatic) multiple myeloma

We hypothesized that increased monoclonal free kappa or lambda immunoglobulin light chains in smoldering multiple myeloma (SMM), as detected by the serum free light chain (FLC) assay, indicates an increased risk of progression to active myeloma. Baseline serum samples obtained within 30 days of diagnosis were available in 273 patients with SMM seen from 1970 to 1995. At a median follow-up of surviving patients of 12.4 years, transformation to active disease has occurred in 59%. The best breakpoint for predicting risk of progression was an FLC ratio of 0.125 or less, or 8 or more (hazard ratio, 2.3; 95% CI, 1.6-3.2). The extent of abnormality of FLC ratio was independent of SMM risk categories defined by number of bone marrow plasma cells (BMPCs) and size of serum M proteins (BMPC>or=10% and serum M protein>or=3 g/dL; BMPC>or=10% but serum M protein<3 g/dL; and serum M protein>or=3 g/dL but BMPC<10%). Incorporating the FLC ratio into the risk model, the 5-year progression rates in high-, intermediate-, and low-risk groups were 76%, 51%, and 25%, respectively. The serum immunoglobulin FLC ratio is an important additional determinant of clinical outcome in patients with SMM.

The Importance of Free Light Chains of Immunoglobulins Determination in Serum

The Importance of Free Light Chains of Immunoglobulins Determination in SerumFor many years, Bence Jones proteinuria has been an important diagnostic marker for multiple myeloma. Relatively new serum tests for free kappa and free lambda light chains of immunoglobulins reflect the production of free light chains more accurately than urine tests. In this study, we examined the value of serum free light chains measurement in the diagnosis of some neoplastic diseases and the discrepance between the findings of serum protein electrophoresis and serum free light chains. Thirty one patients (f=19, m=12) were included in the study, most of them with blood malignant diseases. The results show that in six patients with normal gamma and beta electrophoresis fractions there are abnormal levels of free light chains and/or an abnormal κ/LD ratio. In 20 patients we found an abnormal κ/LD ratio, and in 21 patients we found an abnormal κ or LD level, or both. The obtained results show the important role of serum free light chains determination in identifying patients with monoclonal gammopathies.