Presence Of Monoclonal Immunoglobulin Research Articles

Multiple Myeloma

The second most common hematologic malignancy is the clonal proliferation of neoplastic plasma cells within the bone marrow. There is the presence of monoclonal immunoglobulins in the serum and/or urine. This results in anemia, myelosuppression, bone destruction, and clinical consequences of para-proteinemia on kidney function and other organ systems. The disease manifests through the acronym CRAB (hypercalcemia, renal impairment, anemia, and bone lesions). Less frequent manifestations of multiple myeloma are of extramedullary localizations. Myeloma cells can become independent of the bone marrow microenvironment, circulate freely in the blood, and infiltrate organs. This results in a high-risk state characterized by increased proliferation, evasion of apoptosis, and treatment resistance. It can affect any area of tissue. Most commonly it involves the pleura, lymph nodes, chest wall, liver, skin/soft tissue, lungs, CNS, genitourinary system, breast and pancreas. In patients with confirmed multiple myeloma, the diagnosis of extramedullary involvement is typically established by the presence of pathological soft tissue masses using radiological methods such as computed tomography (CT) scan, positron emission tomography/CT (PET/CT), magnetic resonance imaging (MRI), or ultrasound, along with biopsy or physical examination. The molecular mechanisms underlying the development of extramedullary multiple myeloma (EMM) have not been fully defined. Various cytogenetic abnormalities are observed, and some studies have generated genomic sequencing profiles that distinguish EMM from classic multiple myeloma. While plasma cell leukemia (PCL) and central nervous system (CNS) EMM indicate a poor prognosis, outcomes for other manifestations can be highly heterogeneous. Sensitive imaging modalities including PET/CT and MRI (Fig.1) are integral components of diagnosis and response assessment. Patients with extramedullary multiple myeloma (EMM) have a clear survival disadvantage.

Identifying therapeutic monoclonal antibodies using target protein collision electrophoresis reflex assay to separate the wheat from the chaff

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulins, also known as M-proteins. Therapeutic monoclonal antibodies (t-mAbs) can interfere in laboratory assays used to monitor the state of disease, such as serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). To establish a correct interpretation of IFE, Target protein-Collision Immunofixation Electrophoresis Reflex Assay (T-CIERA) was developed to identify t-mAbs in IFE. Here we demonstrate that T-CIERA is applicable to a wide variety of t-mAbs for which the target protein is commercially available. Moreover, the shift observed was characteristic for each t-mAb, and T-CIERA enabled the identification of multiple t-mAbs sharing a common target protein. Additionally, the lower limit of detection (LLOD) was determined objectively, and T-CIERA demonstrated an adequate LLOD for all tested t-mAbs. Furthermore, T-CIERA was also successfully applied to serum samples obtained from patients receiving daratumumab, isatuximab, elotuzumab, and durvalumab treatment. In conclusion, T-CIERA is a suitable reflex assay for identifying a wide variety of t-mAbs, including those for which no commercial assay is available to deal with their interference. Moreover, CD38-CIERA could serve as an alternative or complementary test to the commercially available Hydrashift assay kits. T-CIERA would enable laboratories without mass spectrometry equipment and expertise in this area to distinguish between drug and disease to improve clinical response monitoring and diagnosis of monoclonal gammopathies.

Investigating the utility of saliva immunoglobulins for the detection of myeloma and using myeloma proteins to clarify partition between oral and systemic immunity.

ObjectivesMyeloma is characterised by the presence of monoclonal immunoglobulin (M‐protein) and the free light chain (FLC) in blood. We investigated whether these M‐proteins and FLC are detectable in myeloma patients’ saliva to evaluate its utility for non‐invasive screening and monitoring of haematological malignancies.MethodsA total of 57 patients with monoclonal gammopathy and 26 age‐matched healthy participants provided paired serum and saliva samples for immunoglobulin characterisation and quantification.ResultsMyeloma patients had IgG or IgA M‐protein levels ranging up to five times and FLC levels up to a thousand times normal levels of polyclonal immunoglobulins. Despite these highly elevated levels, only two IgG and no IgA M‐proteins or FLC could be detected in paired saliva samples. Most patients had reduced levels of serum polyclonal immunoglobulins, but all had normal levels of salivary IgA.ConclusionsImmunoglobulin transfer from blood is not determined by levels in the systemic circulation and more likely dictated by periodontal inflammation and the integrity of the oral epithelium. Immunoglobulins secreted by bone marrow plasma cells do not substantially enter saliva, which represents a poor medium for myeloma diagnosis. These findings, along with normal salivary IgA levels despite systemic immunoparesis, support a strong partitioning of oral from systemic humoral immunity.

A Case Study on Multiple Myeloma

Introduction: Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, anemia, kidney dysfunction, and infections may occur. Multiple myeloma is a hematologic disease characterized by an increase in plasma cells in the bone marrow and, more commonly, the presence of monoclonal immunoglobulin in the blood and / or urine. It is the second major hematologic attack found to have an annual occurrence with an increase in the United States of nearly 15,000 and approximately 45,000, respectively. The condition is higher with age (age between acquisitions of 67 yr). This case report describes a multiple myeloma patient presented with muscle sclerosis and was suspected of having myotonic dystrophy type 2 and illustrates the aspects of differential diagnosis, the use of laboratory and imaging for diagnosis.
 Conclusion: Despite the fact that periodontal illness has a detrimental impact on one's quality of life, those benefits are often ignored. As a consequence, periodontal consultation can be a key component of MM care. Treatment for periodontal disease should begin as soon as possible.

Monoclonal immunoglobulins promote bone loss in multiple myeloma

AbstractMost patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

Diagnosis of haematological diseases with monoclonal gammopathy

AbstractDespite the complexity of lymphoproliferative disorders, the presence of monoclonal immunoglobulins has been providing diagnostic guidance to the haematologist for more than a century. Clinicians may be alerted for the possible presence of serum monoclonal immunoglobulins produced in excess by increased erythrocyte sedimentation rate. Thereafter the diagnosis of monoclonal gammopathy could be proven by serum protein electrophoresis followed by immunofixation to properly identify the M‐protein. In a quarter of the cases, no intact monoclonal immunoglobulin could be detected in the sera, however, free light chains produced in excess may be identified using specialized immunoassays, so called free light chain analysis.Idiopathic monoclonal gammopathy of unknown significance (MGUS) may be present in 1% of the population above age 50 and up to 10% for individuals over 80 years of age. Due to the high frequency of MGUS, between 50‐65% of all monoclonal proteins detected fall into this category and most asymptomatic patients remain undetected for many years. MGUS patients have a 1% per year chance for transformation into malignant monoclonal gammopathies. Presently, it is accepted that most multiple myeloma and amyloidosis patients have their disease developed by transformation from preexisting MGUS. Additionally, some nonmalignant hematological disease also frequently exhibit monoclonal immunoglobulin synthesis, most notably Gaucher’s disease.From the field of neoplastic hematological diseases, smouldering and symptomatic multiple myeloma as well as Waldenström’s macroglobulinaemia almost always exhibit significant amounts of serum M‐protein and/or monoclonal free light chain excess. In case of solitary plasmocytoma, primary AL amyloidosis, cryoglobulinaemia and plasma cell leukemia, the correlation with gammopathy is less strict. Monoclonal gammopathy is less common in different types of non‐Hodgkin lymphoma. Within lymphomas lymphoplasmocytic lymphoma and marginal zone lymphoma more commonly secrete monoclonal immunoglobulins, while other B‐cell lymphomas have 14% cumulative chance for specific gammopathy. In the case of B‐cell chronic lymphocytic leukemia (the most common leukemia), intact monoclonal gammopathy has a 2% appearance, while abnormal free light chains may be detected in a third of the cases. Sporadically monoclonal gammopathy may be detected in chronic myelomonocytic leukemia, refractory anaemia, large granular cell leukemia and angioimmunoblastic lymphoma. Notably, in many of these diseases, the amount of secreted monoclonal immunoglobulin and/or free light chains correlate with the tumor burden and could be used for disease monitoring, especially in indolent hematological neoplasias.

Quantitative Immunoprecipitation Free Light Chain Mass Spectrometry (QIP-FLC-MS) Simplifies Monoclonal Protein Assessment and Provides Added Clinical Value in Systemic AL Amyloidosis

Introduction Early diagnosis, effective therapy and precise monitoring are central for improving clinical outcomes in systemic light chain (AL) amyloidosis. Diagnosis and disease response assessment is primarily based on the presence of monoclonal immunoglobulins and free light chains (FLC). The ideal goal of therapy associated with best outcomes is a complete responses (CR), defined by the absence of serological clonal markers. In both instances, detection of the monoclonal component (M-component) is based on serum FLC assessment together with traditional serum and urine electrophoretic approaches, which present inherent limitations and lack sensitivity particularly in AL where the levels are typically low. Novel mass spectrometry methods provide sensitive, accurate identification of the M-component and may prove instrumental in the timely management of patients with low-level amyloidogenic light chain production. Here we assess the performance of quantitative immunoprecipitation FLC mass spectrometry (QIP-FLC-MS) at diagnosis and during monitoring of AL amyloidosis patients treated with bortezomib-based regimens. Methods We included 46 serial patients with systemic AL amyloidosis diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC). All patients had detailed baseline assessments of organ function and serum FLC measurements. Baseline, +6- and +12-month serum samples were retrospectively analysed by QIP-FLC-MS. Briefly, magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free κ) and free lambda light chains (anti-free λ). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute FLC in monomeric form. Mass spectra were acquired on a MALDI-TOF-MS system (Bruker, GmbH). Results were compared to serum FLC measurements (Freelite®, The Binding Site Group Ltd), as well as electrophoretic assessment of serum and urine proteins (SPE, sIFE, UPE and uIFE). Results Cardiac (37(80%) patients) and renal (31(67%) patients) involvement were most common; 25(54%) patients presented with both. Other organs involved included liver (n=12), soft tissue (n=4), gastrointestinal tract (n=3) and peripheral nervous system (n=2). Baseline Freelite, SPE, sIFE and uIFE measurements identified a monoclonal protein in 42(91%), 22(48%), 34(74%) and 21(46%) patients, respectively. A panel consisting of Freelite + sIFE identified the M-component in 100% of the samples. QIP-FLC-MS alone also identified an M-component in 100% of the samples and was 100% concordant with Freelite for typing the monoclonal FLC (8 kappa, 34 lambda). In 4 patients, QIP-FLC-MS identified an additional M-protein that was not detected by the other techniques. In addition, 4/8(50%) kappa and 4/38(11%) lambda patients showed a glycosylation pattern of monoclonal FLCs at baseline by mass spectrometry. Interestingly, the frequency of renal involvement was significantly lower for patients with non-glycosylated forms (25% vs 76%, p=0.01), while no similar relationship was found for any other organs. During the 1-year follow-up period, 17 patients achieved a CR; QIP-FLC-MS identified serum residual disease in 13(76%) of these patients. Conclusion In our series, QIP-FLC-MS was concordant with current serum methods for identifying the amyloidogenic light chain type and provided, against all other individual tests, improved sensitivity for the detection of the monoclonal protein at diagnosis and during monitoring. The ability to measure the unique molecular mass of each monoclonal protein offers clone-specific tracking over time. Glycosylation of free light chains is over-represented in AL patients which may allow earlier diagnosis and better risk-assessment of organ involvement. Persistence of QIP-FLC-MS positive M component in patients otherwise in CR may allow targeted therapy. Overall, QIP-FLC-MS demonstrates potential to be exploited as a single serum test for precise serial assessment of monoclonal proteins in patients with AL amyloidosis. Disclosures Wechalekar: GSK: Honoraria; Janssen-Cilag: Honoraria; Amgen: Research Funding; Takeda: Honoraria; Celgene: Honoraria.

Glycosylationof Immunoglobulins Determine Bone Loss in Multiple Myeloma

About 80% of patients with multiple myeloma develop a severe osteolytic bone disease causing pain and fractures. The myeloma cells secrete immunoglobulins and the presence of monoclonal immunoglobulins in serum is a hallmark of the disease. Immunoglobulins play a role in bone loss, but have not been linked with the bone disease in multiple myeloma. In this work, we isolated immunoglobulins from serum of myeloma patients using protein G coupled magnetic beads and columns. We found that immunoglobulins from patients with bone disease (n=16) promoted osteoclast differentiation when added to human monocyte-derived pre-osteoclasts (p=0.006). We next fractionated the immunoglobulin samples by size-exclusion chromatography and found that the "osteoclast promoting activity" was in the high-molecular weight fractions, suggesting that they are in complexes. Since extent of complex formation may be determined by glycosylation, we examined whether there is a difference in immunoglobulin glycosylation between healthy controls and patients, and whether it changes during disease progression. To this end we analysed IgG glycosylation in serum samples from patients (n=72) and age and sex matched controls (n=51). These analyses showed that patient IgG was less galactosylated (p=0.02) and less sialylated (p=0.04) compared with control IgG. Moreover, patients with bone disease (n=43) had significantly less galactose on IgG compared with patients without bone disease (p=0.02, n=33). Supporting this data, we found that galactosidase treatment of immunoglobulins from patients without bone disease induced osteoclastogenesis (p=0.03), whereas addition of galactose to immunoglobulins of patients with bone disease removed their pro-osteoclastogenic effect (p=0.01). Further, the glycosyltransferases ST6GAL1 and B4GALT11, which add sialic acid and galactose to the sugar chain, respectively, are less expressed in plasma cells obtained from patients with bone disease (n=137) compared with those without (n=36, p<0.002, p<0.001, GSE755). Importantly, we observed a significant reduction of IgG glycosylation (p=0.02, n=8) in serum samples obtained from individual patients before and after the onset of bone disease. Taken together, our data support that immunoglobulins promote bone loss in multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Polyclonal immunoglobulin recovery after autologous stem cell transplantation is an independent prognosis factor for survival in patients with multiple myeloma

Multiple myeloma (MM) is a plasma cell neoplasm characterized by the presence of monoclonal immunoglobulin (Ig) in serum and/or urine and clinical symptoms related to the CRAB features and myeloma-defining events. MM is a clinically and cytogenetically heterogeneous disease and survival outcome varies considerably depending on the risk status and treatment of each patient. Until now, treatment strategy for MM has been evolving rapidly by the introduction of several new classes of agents such as proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies.

Scleredema associated with multiple myeloma or MGUS: treatment report of four cases

Scleredema is rare scleroderma-like fibromucinosis characterized by progressive thickening of the skin caused by excessive collagen and mucin depositions in the dermis, these disease may have a serious clinical course with organ involvement. In majority of scleredema cases, presence of monoclonal immunoglobulin (MIG) was detected. Various treatment regimens have been proposed, however with inconsistent outcomes. Here, we present our treatment experiences with four cases of scleredema associated with paraproteinemia.

P07.06 * A CASE OF BING-NEEL SYNDROME PRESENTING AS EPIDURAL METASTASIS

In 2005, a 63 year-old man with prior hypertension, started to complain persistent asthenia and weight loss. Blood tests revealed anemia and hypergammaglobulinemia. Immunoelectrophoresis confirmed the presence of monoclonal immunoglobulin M (IgM) with kappa light chain (34 g/L) in the serum. Multiple retroperitoneal lymphoadenopathies were detected. Bone marrow examination disclosed lymphoplasmocytic proliferation with small lymphocytes and plasmacytoid lymphocytes. The patient received 6 cycles of fludarabine, cyclophosphamide and rituximab from November 2005 to July 2006 with partial remission. Maintenance therapy with monthly administration of rituximab was then performed from October 2006 to January 2007. In July 2011 a relapse occured and chemotheraphy with 2-CdA cladribine and rituximab was introduced with complete response. In January 2014 the patient came to our attention complaining a 2 month hystory of difficulties in walking, stifness in the legs, hand clumsiness and episodic urinary urgency. Neurological examination showed mild paraparesis, reduced proprioception in the legs without sensory levels, diffuse hyper-reflexia, bilateral Babinski sign and increased tone in the legs. Serum protein electrophoresis and immunofixation confirmed a gamma band and monoclonal Ig M kappa light chains. Cerebrospinal fluid (CSF) examination revealed 96 cells/mm3 with 90% of lymphocytes, low glucose concentration (31 mg/dL) and increased protein level (1288 mg/dl). CSF cytology and flow cytometry proved negative for malignancy, there was T cell CD5+ predominance in the cells seen. Magnetic resonance imaging (MRI) of the brain showed leptomeningeal enhancement along the brainstem. MRI of the spinal cord showed an enhancing epidural mass spanning levels C2-C4 causing spinal cord compression and associated with mild hyperintensity in the cervical cord on T2-weighted sequences at this level, moreover leptomeningeal enahncement along inferior cervical and upper dorsal spinal cord was present. The patient underwent a decompressive laminectomy at C2-C4. Postoperative course was regular, without complications. Pathological analysis of tissue fragments from the epidural lesion revealed an atypical, diffuse lymphoid infiltrate. Immunohistochemical staining of these cells was positive for CD20, CD79 and partially for CD38, negative for CD10, CD5, CD3, CD23, ciclin D1. Staining for immunoglobulin light chains revealed kappa light chain. These results support the diagnosis of epidural localization of WM. Even if epidural infiltration can occur in lymphoid neoplasms such as Hodgkin's and non Hodgkin's lymphoma, plasmocytoma and multiple myeloma, this has been exceptionally reported in WM (Al-Halabi H, Roberge D American Journal of Hematology 2006, 81: 955-958). In our case pathological analysis of a biopsy specimen confirmed an epidural localization of WM.

The Significance Of Abnormal Plasma Cell Clone In Bone Marrow Of Primary Systemic Light Chain Amyloidosis Patients

In this study we analyzed the immunophenotype characteristics of the plasma cells and evaluated the significance of the abnormal plasma cell clone in bone marrow in primary systemic light chain amyloidosis (AL) patients. Fresh bone marrow samples were collected from 74 cases of plasma cell disease (PCD), including 51 cases of AL, 21 cases of multiple myeloma (MM), 2 cases of Waldenström's macroglobulinemia (WM). All patients diagnosed according to WHO 2008 diagnostic criteria. The diagnosis of AL was confirmed by the presence of monoclonal immunoglobulin or free light chain in blood or urine, and/or amyloidosis in fat tissues or biopsies by Congo red staining. Ten healthy donors were also collected as controls. Their clinical characteristics and immunophenotype of bone marrow cells were compared and analyzed. The immunophenotype were analyzed with a panel of antibodies including CD45, CD38, CD138, CD117, CD56, CD19, CD20, Igκ, Igλ, CD7, CD22, CD3, CD34 and CD27 by flow cytometry (FCM). The results were for statistical processing.The prominent feature of AL patients was multi-organ and multi-system involvement. Kidney was the major organ involvement (82.4%), followed by cardiovascular system (58.8%); MM mainly had the clinical manifestations of bone lesions (85.7%) and renal involvement (47.6%). The serum immunoglobulin of AL mainly manifested as λ light chain (74.5%), while the majority of MM manifested as κ (61.9%). In the 51 patients of AL, the ratio of plasma cellsin bone marrow was mean 3.87% (0.17∼9.34%) by FCM, and 4.47% (0∼14.5%) by morphological examination. In MM, the ratio of plasma cells was mean 13.17% (1.30∼48.91%) by FCM and 33.55% (3.0∼81.5%) by morphological examination. The plasma cells proportion between AL and MM had significant difference (P< 0.05). The κ or λ light chain restriction can be used for the detection of abnormal plasma cell clones in AL patients. The κ/λ ratio>4.0 or <0.5 can be used as the criteria to identify light chain restriction in plasma cells in AL patients. The 31/51 cases of AL could detected abnormal plasma cell clone that used κ/λ light chain restriction and were mainly expressed λ light chain (24/31, 77.4%). The 21 cases of MM had light chain restriction, mainly expressed κ light chain (13/21, 61.9%) (P<0.05). In CD45/SSC scattergram, the position of abnormal plasma cells of AL patients varied in a wider range. According to the features of CD38+/CD138+ as the basic markers for plasma cells, abnormal plasma cells were CD45 negative or weak positive in AL patients, similar to the CD45 level distribution in malignant plasma cells in MM. In WM, the proliferated cells were plasmacytoid lymphocytes with CD45 weakly or strong positive. FCM can identify abnormal plasma cell clone in bone marrow of AL patients. In 51cases of AL, 78.4% of bone marrow plasma cells were CD56+, 68.6% were CD117+, and 88.2% were CD19-. In 21 of MM, 66.7% were CD56+, 38.1% were CD117+, and 90.4% were CD19-. These results manifested significant difference compared with those of normal plasma cells (P< 0.05). In 2 cases of WM, these plasmacytoid lymphocytes were CD19+ and CD56-, CD117-.The ratios of CD56+, CD117+, CD19-, and CD45-/dim in bone marrow plasma cells were significantly higher in AL patients than in WM patients and healthy individuals (P<0.05), but were similar to those in MM patients (P>0.05). The main difference between AL and MM was the larger size of plasma cell group in MM (P＜0.05).In summary, according to light chain restricted expression and abnormal immunephenotype by FCM analysis we can determine abnormal plasma cell clone in bone marrow of AL patients and the abnormal plasma cells clone can be used as an important diagnostic marker of AL. Disclosures:No relevant conflicts of interest to declare.

Anti-myelin-associated glycoprotein polyneuropathy coexistent with CREST syndrome

Clinical involvement of the peripheral nervous system in the calcinosis cutis, raynaud's phenomenon, esophageal dismotility, sclerodactyly and telangiectasia (CREST) variant of systemic sclerosis occurs infrequently and is characterized by axonal degeneration due to necrotizing vasculitis. We report a female patient with a known history of CREST syndrome, which developed a slowly progressive, distal symmetric demyelinating sensorimotor polyneuropathy (PN), with tremor and ataxia as prominent features, compatible with anti-myelin associated glycoprotein (MAG) PN. The diagnosis of PN was established by the presence of monoclonal immunoglobulin M anti-MAG antibodies (Thin-Layer Chromatography, Western Blot and enzyme-linked immunoabsorbent assay). Given the evidence that in CREST activation of T-helper cells is observed and that anti-MAG antibodies, despite the fact that they are T-cell-independent, may be influenced by an increase in T-helper function, the coexistence of these two rare autoimmune disorders in the same patient may not be incidental but related to the underlying immunological mechanisms involved.

Bone marrow plasma cell separation – validation of separation algorithm

Monoclonal gammopathy is characterized by the presence of monoclonal immunoglobulin which is produced by a specifi c clone of terminally differentiated long-living plasma cells (PC) in the bone marrow. More detailed understanding of this clone at the genomic and proteomic level as well as its comparison with the population of normal PC is possible only after the clone is separated from other BM cells.

Jejunal Amyloidosis: A Rare Cause of Severe Gastrointestinal Bleeding

1Department of Medicine; 2Division of Pathology; 3Division of Gastroenterology, University of British Columbia, Royal Columbian Hospital, New Westminster, British Columbia Correspondence: Dr Justin Cheung, Department of Gastroenterology, Royal Columbian Hospital, 410-301 East Columbia Street, New Westminster, British Columbia V3L 3W5. Telephone 604-525-0155, fax 604-525-1699, e-mail justa48@hotmail.com Received for publication December 22, 2011. Accepted December 29, 2011 CASE PRESENTATION An 81-year-old Jamaican woman presented with melena and severe anemia, and a hemoglobin level of 47 g/L (normal range 115 g/L to 160 g/L). A gastroscopy and colonoscopy were negative for any lesions. Duodenal biopsies were normal. A subsequent computed tomography scan of the abdomen with contrast showed diffuse thickening from the jejunum to the distal small bowel and a small area of active bleeding in the mid small bowel. A technetium-99m red blood cell scan demonstrated active bleeding in the proximal jejunum. Push enteroscopy (120 cm to 150 cm below incisor) revealed mild diffuse friability starting from the proximal jejunum, as well as erosions, nodularities, polypoid protrusions and valvulae conniventes thickening in the remaining jejunum (Figures 1A and 1B). Jejunal biopsies showed extensive amyloid deposition in the lamina propria and submucosa, with Congo red stain demonstrating apple-green birefringence under polarized light (Figure 2), and hematoxylin counterstain revealing amorphous, salmon-pink, sparsely cellular hyaline material (Figure 3). Serum protein electrophoresis, immunofixation and a serum-free light chain assay confirmed the presence of monoclonal immunoglobulin G lambda paraprotein. The patient was transfused with packed red blood cells as needed (average one unit per day), and treated with bortezomib and dexamethasone for primary amyloidosis. By day 17 of hospitalization, the bleeding had resolved and the patient was subsequently discharged without any further bleeding episodes. DISCUSSION Amyloidosis is a multisystem disease characterized by extracellular deposition of abnormal protein fibrils in various tissues and organs. The protein fibrils are derived from monoclonal immunoglobulin light chains in primary amyloidosis (AL), acute phase reactant serum amyloid A in secondary amyloidosis (AA), and beta2-microglobulin in dialysis-related amyloidosis (Ab2m). Clinical manifestations in amyloidosis are variable, but the heart and kidneys are most commonly involved. Gastrointestinal symptoms due to bleeding, dysmotility and malabsorption are present in up to 60% of AA (1), but in only 1% of AL (2). The diagnosis of amyloidosis can only be confirmed by tissue biopsy because endoscopic and radiologic findings are nonspecific. Therapy is directed at the underlying cause in AL (plasma cell dyscrasia) and AA (infection, inflammation and malignancy), whereas treatment in Ab2m involves alteration of dialysis mode or consideration of renal transplantation (3). Severe gastrointestinal bleeding rarely occurs as the sole or initial manifestation of systemic amyloidosis (4). Mechanisms by which hemorrhage occurs in gastrointestinal amyloidosis have been postulated, including intestinal ischemia, necrotic ulceration, mucosal tearing, vascular fragility, abnormal platelet aggregation, and factor IX and X deficiencies (5,6). In addition to supportive care for bleeding, treatment must be tailored to the specific type of amyloidosis, as determined by endoscopy and histology. Endoscopy classically shows polypoid protrusions and valvulae conniventes thickening in AL; a imAge of the month

Transient inflammatory reaction during lenalidomide plus reduced-dose dexamethasone therapy in two patients with relapsed multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by the presence of monoclonal immunoglobulin and related end-organ damages [1]. Lenalidomide is one of the most potent immunomodulatory drugs (IMiDs) that induce significant anti-tumor effects in patients with newly diagnosed and refractory MM [2–5]. This agent also has pleiotropic properties including immunomodulatory and anti-inflammatory effects, and has been shown to stimulate T cells and NK cells to produce Th1 type cytokines such as interleukin (IL)-2 and interferon-c, while displaying inhibitory effects on the production of Th2 type cytokines and pro-inflammatory cytokines such as IL-4, IL-6, and tumor necrosis factor a (TNF-a) in vitro [6]. The clinical relevance of such immunomodulatory effects is yet to be elucidated in patients with MM. Here, we report two patients with relapsed MM who showed transient inflammatory reaction during lenalidomide plus reduced-dose dexamethasone therapy. Case 1 A 49-year-old male was suffering from MM (IgGj type) in stage IIA and international staging system (ISS) 2. He was treated with three courses of vincristine, adriamycin, and dexamethasone (VAD); however, his disease activity was not improved. He received two courses of BD therapy (bortezomib 1.3 mg/m on days 1, 4, 8, and 11, and dexamethasone 20 mg on days 1, 2, 4, 5, 8, 9, 11, and 12), and achieved very good partial response (VGPR). One month later, he experienced grade 3 peripheral neuropathy and further BD therapy was discontinued. Peripheral blood stem cells (PBSC) were harvested, but he was followed-up without transplantation due to persistent peripheral neuropathy. Two years later, serum IgG levels were gradually increased, and he was referred to our hospital. Laboratory examination revealed that Hb level was 14.0 g/dl and serum total protein 10.0 g/dl with 36.3% M-protein (3.63 g/dl). Bone marrow was normocellular with 13.2% MM cells. As salvage therapy, he received lenalidomide (25 mg on days 1–21) plus dexamethasone (20 mg on days 2, 9, and 16) therapy on a 28-day cycle, but the dosage and frequency of dexamethasone was reduced because of hypertension and skin acne (Fig. 1a). On day 8 at cycle 1, serum CRP level was suddenly increased to 6.32 mg/dl and decreased to 0.85 mg/dl on day 12 after administration of dexamethasone, but re-elevated to 8.37 mg/dl on day 15. Plasma fibrinogen level was also increased up to 916 mg/dl, but white blood cell count remained normal range. He experienced grade 1 dry skin and fatigue as adverse events, but there were no signs and symptoms of infection or inflammation such as fever and arthralgia. We carefully continued the treatment, and serum CRP and plasma fibrinogen levels became unremarkable during subsequent therapeutic cycles. His serum total protein was gradually decreased to 6.7 g/dl with 11.2% M-protein (0.75 g/dl) achieving PR after 4 cycles of the therapy. Case 2 A 58-year-old male was admitted to our hospital because of compression fracture in lumbar vertebrae. He S. Ozaki (&) T. Harada S. Fujii S. Nakamura H. Miki A. Nakano K. Kagawa K. Takeuchi M. Abe T. Matsumoto Department of Medicine and Bioregulatory Sciences, The University of Tokushima Graduate School of Health Biosciences, 3-18-15 Kuramoto, Tokushima 770-8503, Japan e-mail: ozakishu@clin.med.tokushima-u.ac.jp

Laboratory Characterizations on 2007 Cases of Monoclonal Gammopathies in East China

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor beta1 (TGFbeta1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFbeta1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonal immunoglobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain kappa 6.1%, lambda 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (kappa/lambda ) 94.4%, quantitation of Igs 83%, light chain quantitation 80.9%, and urine light chain ratio (kappa/lambda) 58.0%. Plasma TGFbeta1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C>T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitation and urine light chain determination. Although TGFbeta1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFbeta1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study.

A Case of Type I Cryoglobulinemia Associated with a Monoclonal Gammopathy of Undetermined Significance (MGUS)

A 79-year-old Japanese male who had been suffering from renal insufficiency of unknown causes visited us with reddish papules and purpura on both forearms of a month's duration. A skin biopsy was performed from the right forearm, and the histopathology showed an obstruction of superficial blood vessels with eosinophilic amorphous materials suggesting cryoglobulins. Although further investigations revealed the presence of monoclonal immunoglobulin (Ig) G-kappa type I cryoglobulin, a bone marrow biopsy demonstrated that there was 7.6% proliferation of plasmacytoid cells, and the serum level of Ig G was less than 3 g/dl. The diagnosis was type I cryoglobulinemia associated with monoclonal gammopathy of undetermined significance as the possible cause of chronic renal failure. Therefore, purpuric changes on extremities of the elderly, especially those complaining of renal failure should, be taken seriously and not regarded simply as an age-related phenomenon.

Correlation of Serum Free Light Chains and Bone Marrow Plasma Cell Infiltration in Multiple Myeloma.

Diagnostic criteria for multiple myeloma include abnormal plasma cell infiltration of the bone marrow plus the presence of monoclonal immunoglobulin in the serum and/or monoclonal free light chains in the urine. However, recent studies have indicated that measurement of free light chains in the serum (sFLC) is more sensitive than urine assays. Also, because of the slow clearance of IgG from serum (half-life ~20 days compared with 2–6 hours for sFLC) intact immunoglobulin assays can be slow to reflect the full extent of response to treatment. The aim of this study was to compare the relative sensitivity and specificity of serum free light chain (sFLC) measurement, urine free light chain measurement (uFLC) and serum immunofixation (sIFE) with bone marrow analysis for assessment of myeloma. All 45 patients studied were enrolled in the UK MRC Myeloma VII trial and 75 serum samples were selected for sFLC measurement and sIFE. The sera had been collected at various times before, during and after treatment but all within 4 days of a bone marrow assessment and uFLC measurement (by radial immunodiffusion assay). sFLC results were classified as abnormal if the kappa/lambda ratio was outside the normal range and for uFLC, if there was >40mg/L. The bone marrow assessment was called abnormal if 5% or greater plasma cell infiltration was recorded in aspirate or trephine, in accordance with the EBMT criteria. The results of the comparisons are summarised in the table.Summary of paraprotein assaysBone MarrowNormalAbnormalSerum Free Light ChainsNormal194Abnormal547Urine Free Light ChainsNormal2121Abnormal330Serum ImmunofixationNormal105Abnormal1446Of the three paraprotein assays, sFLC had the highest concordance with bone marrow biopsy. Compared with the bone marrow assessment, the relative sensitivity and specificity of the sFLC assays was 92% and 79% respectively. For uFLC the values were 59% and 88%, respectively and for sIFE, 90% and 42% respectively. Of the 5 sera abnormal by the sFLC assays but with concurrent normal bone marrow results, all were abnormal by sIFE. Of the 4 patients with normal sFLC results but abnormal marrows, 3 were sIFE positive and of the 5 with negative sIFE results but abnormal marrows, 4 had abnormal sFLC ratios. Only 1 patient had an abnormal marrow with normal sFLC and sIFE results. In this comparison sFLC measurement showed a good degree of correlation with bone marrow assessments of myeloma, while uFLC assays were considerably less sensitive. Both sFLC and sIFE assays appeared to identify disease in 5 patients who had normal bone marrow assessments. This was presumably because the serum assays sample monoclonal protein produced throughout the body while distribution of the disease in the bone marrow is occasionally “patchy”. The sIFE results were positive in a much higher number (14) of bone marrow-negative patients. The 9 “extra” positives, which had normal bone marrow results and free light chain ratios, might result from a greater sensitivity for detecting tumours producing intact immunoglobulin with low levels of free light chains. Some of the 9 subsequently became sIFE negative so the slow clearance of monoclonal intact immunoglobulin is an alternative explanation for the discordant results. This could not be proven, however, as all patients had some form of treatment in the intervening period.

The Classification and Typing of Amyloid Deposits

The diagnosis of amyloidosis, unlike many other diagnoses that fall on the shoulders of the pathologist, is based on a simple tinctorial property. Binding of the cotton wool dye Congo red, demonstrating green birefringence when viewed under polarized light, fulfills all diagnostic criteria for amyloid. Reporting amyloidosis in a pathologic specimen is reminiscent of a histologic diagnosis of “lymphoma.” The demonstration of amyloid in and of itself is not particularly helpful to the clinician, and an accurate determination of the type of amyloid is just as important as recognizing the presence of amyloid. The clinician will have many questions for the pathologist. Is the amyloid systemic or localized? Is the amyloidosis primary, secondary, or inherited? The former question is important because the majority of patients with localized disease do not require systemic therapy and the long-term prognosis is excellent. The latter question is important because the prognoses for the 3 forms of systemic amyloid and the treatment of the 3 disorders are vastly different. One often involves chemotherapy and stem cell transplantation; another might lead to liver transplantation. Accurate classification becomes imperative. Some forms of localized amyloid are light chain–derived, but the light chain production is local and not part of a systemic plasma cell dyscrasia.1 Localized forms of amyloid might be suspected because of the organ involved. When deposits of amyloid are found in the bladder, urethra, and ureter, it is virtually always a localized process. Most cases of amyloid in the skin and conjunctivae are localized. Amyloidosis of the larynx and tracheobronchial tree is not a systemic disorder, although it may cause troublesome complications. Amyloid deposits in the atria of the heart, pleura, and the articular cartilage generally are localized, and the diagnosis of systemic amyloidosis should not be made unless there is other evidence of visceral involvement. The majority of patients seen in the United States with systemic amyloid have immunoglobulin light chain amyloid (AL) or primary type. The origin of the fibril is an immunoglobulin light chain or immunoglobulin heavy chain fragment. Virtually all patients have a demonstrable clonal plasma cell dyscrasia, and this fact can be used to advantage in classifying the amyloid. These patients will have a detectable monoclonal light chain in the serum or urine shown by immunofixation or an elevation of the immunoglobulin free light chain level revealed by nephelometry.2 Bone marrow examination will demonstrate clonal plasma cells as the source of the light chain production.3 The finding of a monoclonal protein is helpful, but it is not sufficient to diagnose AL. Occasionally a patient will have an incidental monoclonal gammopathy of undetermined significance and a form of amyloid that is not immunoglobulin light chain in origin.4 Finding amyloid deposits on biopsy in the presence of monoclonal immunoglobulins is strong but not conclusive evidence of AL. Clinically, it is difficult to distinguish primary, secondary, familial, and senile forms of amyloid from one another. In the Mayo Clinic (Rochester, MN) experience, half of the patients with inherited amyloidosis do not have a positive family history. Patients with secondary amyloidosis typically have nephrotic syndrome. Renal biopsies demonstrate amyloid deposits. These patients also might have significant gastrointestinal involvement. Patients with hereditary systemic amyloidosis can have renal involvement