Kappa Light Chain Research Articles

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

A New Laboratory Workflow Integrating the Free Light Chains Kappa Quotient into Routine CSF Analysis.

We performed this cohort study to test whether further analysis of intrathecal inflammation can be omitted if the free light chain kappa (FLCκ) quotient is within the reference range in the corresponding quotient diagram. FLCκ concentrations were measured in serum and cerebrospinal fluid (CSF) samples. The intrathecal fraction (IF) of FLCκ was calculated in relation to the hyperbolic reference range. 679 patient samples were used as a discovery cohort (DC). The sensitivity and negative predictive value (NPV) of the FLCκ-IF for the detection of an intrathecal humoral immune response (CSF-specific OCB and/or IF IgG/A/M > 0%) was determined. Based on these data, a diagnostic algorithm was developed and prospectively validated in an independent validation cohort (VC, n = 278). The sensitivity of the FLCκ-IF was 98% in the DC and 97% in the VC with a corresponding NPV of 99%. The use of the FLCκ-IF as a first line analysis would have reduced the Ig and OCB analysis by 62% in the DC and 74% in the VC. The absence of a FLCκ-IF predicts the absence of a humoral intrathecal immune response with a very high NPV of 99%. Thus, integration of our proposed algorithm into routine CSF laboratory analysis could help to reduce analytical efforts.

CCN1 upregulates IL-36 via AKT/NF-κB and ERK/CEBP β-mediated signaling pathways in psoriasis-like models.

Psoriasis is a chronic skin disorder characterized by epidermal keratinocyte hyperproliferation and inflammatory infiltration. CCN1 (also termed CYR61 or cysteine-rich angiogenic inducer 61) is an extracellular matrix-associated protein that is involved in multiple physiological functions. In psoriasis, we recently demonstrated that the overexpression of CCN1 promoted keratinocyte proliferation and activation. Furthermore, CCN1 was highly expressed in psoriatic skin lesions from psoriasis vulgaris patients. Here, we dissect the underlying molecular mechanism in imiquimod (IMQ) and interleukin (IL)-23-induced psoriasis-like models. Our results demonstrate that CCN1 can significantly upregulate IL-36 production in the murine skin of IMQ and IL-23-induced psoriasis-like models. Injection of CCN1-neutralizing antibody improved epidermal acanthosis and significantly reduced IL-36 production in vivo. These results suggest that CCN1 can be a critical upstream pro-inflammatory factor in psoriasis. In primary normal human epidermal keratinocytes, we demonstrated that CCN1 can selectively induced the production of IL-36α and IL-36γ through the activation of the protein kinase B (AKT)/nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and extracellular-regulated kinase (ERK)/CCAAT/enhancer binding protein β (CEBPβ) signaling pathways via integrin receptor α6β1 in vitro. Our results suggest that targeting CCN1 can be a potential therapeutic strategy for psoriasis.

Unusual Diagnosis of Multiple Small Bowel Masses

Question: A 76-year-old man with hypertension and well-controlled diabetes presented to the hospital with poor appetite, abdominal distention, and intermittent nausea and vomiting for several weeks. He reported no fevers, sick contacts, or recent travel, but had an 18-pound unintentional weight loss during this time period with inability to tolerate solids for 4 weeks. Physical examination was notable for abdominal distension and tympany to percussion. Blood work showed a white blood cell count of 4,200/μL with a normal differential, hemoglobin of 13.1 g/dL, hematocrit of 38.2%, red blood cell count of 4.5 million/μL, and a platelet count of 239,000/μL. The metabolic profile was only notable for a potassium level of 2.8 mmol/L. There was no elevation in creatinine, calcium, or alkaline phosphatase. A computed tomography (CT) abdomen/pelvis scan without contrast showed multiple small bowel masses resulting in a small bowel obstruction, with the most proximal small bowel mass located in the third portion of the duodenum (Figure A). After NG tube decompression, enteroscopy was performed and identified an ulcerated mass near the ampulla measuring approximately 4 cm, and a single 1.5-cm ulcerated mass with adjacent edematous and friable mucosa in the third portion of the duodenum (Figure B). Duodenal biopsy showed small intestinal mucosa with prominent lymphoplasmacytic infiltrates in the lamina propria. Notably, these cells stained positive for CD138 on immunohistochemistry (Figure C). Serum protein electrophoresis and urine protein electrophoresis did not reveal any significant monoclonal spike or paraproteins. A subsequent bone marrow biopsy from the iliac crest was notable for an increased number of abnormal plasma cells, with less condensed nucleus and nucleolus. These abnormal plasma cells constituted 7% of the marrow (reference range, 0%–5%). The rest of the marrow differential consisted of the following: 63% myeloid cells (reference range, 30%–67%), 5% lymphocytes (reference range, 3%–17%), 25% erythroid cells (reference range, 10%–35%), and zero blasts (reference range, 0.3%–5%). There was no expression of kappa or lambda light chain. A subsequent whole-body positron emission tomography (PET)/CT showed no lytic lesions.

A novel and robust approach to defining population-based reference ranges for a serum free light chain assay in a clinical laboratory

Abstract Serum protein electrophoresis and immunofixation are used to detect the presence of monoclonal heavy and light immunoglobulin chains for the diagnosis of plasma cell neoplasms (PCN). In conjunction, the Freelite® assay has been used to detect excess free kappa and lambda light chains produced by PCN, where the kappa:lambda free light chain ratio (KLR) serves as a surrogate marker of clonality and disease. The manufacturer-defined KLR reference range of 0.26-1.65 is used in internationally recognized criteria for PCN diagnosis and monitoring, and KLR results affect clinical decision making, disease categorization, and response assessment. Our laboratory has verified the manufacturer-defined Freelite® KLR reference range three times using healthy control sera since assay implementation in 2006. Despite this, high percentages of abnormal KLR have consistently been observed among immunofixation-defined monoclonal-negative specimens. We therefore retrospectively interrogated 48,012 serum free light chain clinical results reported at our institution between 2010-2020 by extracting results from our laboratory information system and analyzing population distributions using the R programming language. For all specimens, the distribution of KLR values was shifted towards the towards the upper limit of the manufacturer-defined reference range, indicating that it poorly describes both our monoclonal-negative and monoclonal-positive patient populations. Furthermore, &amp;gt;80% of monoclonal-negative specimens exhibited KLR above the median of the manufacturer-defined reference range (0.26-1.65), while 24% (1,226/5,057) unexpectedly exhibited KLR &amp;gt;1.65. We found that this phenomenon has been present consistently since 2010 and is not affected by patient sex or age, impaired renal function, lot-to-lot variation, or assay drift. We therefore sought to establish an institution-appropriate KLR reference range by defining a reference population from within our existing dataset. We leveraged International Classification of Diseases codes, estimated glomerular function data, and serum/urine immunofixation results to exclude specimens from individuals with PCN- or renal-related diagnoses, impaired renal function, and prior, current, or subsequent monoclonal components. The resultant 1,536 monoclonal-negative reference specimens were then classified according to the manufacturer-defined KLR reference range, where 13% remained abnormally elevated, consistent with the published false positive rate in healthy volunteers. Lastly, we determined a new KLR reference range of 0.66-2.21 using the central 95% of KLR values from these reference specimens. When applied to our dataset, this institution-specific KLR reference range improved the accurate classification of specimens containing monoclonal free light chains by immunofixation, while reducing the false-positive rate in monoclonal-negative specimens. Together, our findings support the use of historical KLR and immunofixation data from routine clinical testing to derive institution-appropriate KLR reference ranges, which can then be used for continuous quality improvement. Implementation of this more-accurate KLR reference range should improve subsequent test utilization by non-specialist providers, reduce concern for/over-diagnosis of kappa-involved disease, and improve the detection and monitoring of lambda-involved disease.

A Rare Case of Amyloid Light Chain Amyloidosis with Extensive Hepatic Involvement

Abstract Introduction/Objective Amyloid light chain (AL) amyloidosis is a life-threatening disease entity, with an incidence of 10–15 per million per year in the USA. Amyloidosis is caused by the extracellular deposition of insoluble fibrillary amyloid protein resulting in multiorgan damage. Though amyloidosis is primarily a systemic disease, 10%-20% of cases can be localized. Methods/Case Report We present a case of a 37-year-old male admitted with predominant hepatic manifestations, whose liver biopsy led to the diagnosis of amyloidosis. The patient presented with intermittent abdominal pain, weight loss, persistent hypoglycemia, hepatomegaly, and raised liver enzymes. Imaging studies showed hepatomegaly with a heterogeneous lace like appearance of the liver parenchyma. Liver biopsy revealed an extensive deposition of amyloid protein in peri-sinusoidal spaces. The amyloid protein is highlighted by crystal violet stain and revealed apple- green birefringence on polarized microscopy after Congo red staining. On further evaluation, elevated kappa and lambda free light chains with increased kappa-lambda ratio were identified by serum protein electrophoresis. Subsequent bone marrow aspirate and biopsy identified a monoclonal kappa positive plasma cell neoplasm with an amorphous eosinophilic infiltrate. Liquid chromatography-tandem mass spectrometry on bone marrow biopsy detected a peptide profile consistent with AL (kappa) type amyloid deposition. Results (if a Case Study enter NA) NA. Conclusion AL amyloidosis patients with predominant hepatic involvement have a worse prognosis. Rapid diagnosis and prompt clinical intervention prevents further organ damage. This case study aims to promote awareness of the unusual presentations of amyloidosis.

A Case of ALECT2 Renal Amyloidosis Associated with IgG4 Related Kidney Disease, Membranous Nephropathy and Early Diabetic Kidney Injury

Abstract Introduction/Objective ALECT-2 amyloidosis is a rare type of amyloidosis that mostly involves kidneys with other organs rarely affected. It has a high prevalence among patients of Hispanic descent. Membranous nephropathy is one of the most common causes of proteinuria in adults. IgG4-related disease is a systemic disease, which commonly involves the pancreas, but occasionally affects the kidney and manifests as chronic renal insufficiency. Here we describe a very unusual case of concurrence of membranous nephropathy, IgG4 disease involving the kidney, ALECT2 amyloidosis, and early diabetic kidney injury. Methods/Case Report A 49-year-old Hispanic male patient with a history of diabetes and IgG4-related autoimmune pancreatitis and primary sclerosing cholangitis presented with abrupt onset of proteinuria and hypoalbuminemia. A kidney biopsy was performed and showed severe interstitial plasma cell-rich inflammatory infiltrates and interstitial fibrosis which had a storiform pattern. The glomerular basement membranes (GBM) showed focal pinpoint holes but no spikes by silver stain. Immunofluorescence microscopy (IF) showed diffuse and finely granular capillary loop staining for IgG, with Kappa and lambda light chains of equal intensity. IF for Anti-phospholipase A2 receptor (PLA2R) was negative. Immunohistochemical (IHC) stain showed IgG4 positivity in about 60% of IgG-positive plasma cells. Congo red was positive for birefringent deposits predominantly in the interstitium and arteriolar walls with focal deposits in the glomerular mesangium and capillary wall. IHC stain for Amyloid AA and DNAJB9 were negative. Electron microscopy showed scattered subepithelial immune complex-type electron dense deposits consistent with membranous nephropathy, randomly oriented fibrils in interstitium, mesangium and GBM, consistent with amyloidosis, and thickening of GBM (average 559 nm), consistent with early diabetic kidney change. The tissue was sent for mass spectrometry which showed a peptide profile consistent with ALECT-2 (Leukocyte chemotactic factor 2) type amyloidosis. Results (if a Case Study enter NA) NA Conclusion In up to a third of cases reported in the literature, a concomitant renal pathology was present. Diabetic nephropathy was the most common concurrent pathology, to be followed by IgA nephropathy and membranous nephropathy. However, the concurrence of membranous nephropathy, IgG4 disease involving the kidney, ALECT2 amyloidosis, and early diabetic kidney injury has never been described before.

Extranodal Marginal Zone Lymphoma of the Thyroid with Extensive Amyloid Deposition: A Rare Case Presentation

Abstract Introduction/Objective Extranodal marginal zone lymphoma (MZL) is a rare indolent tumor with the potential of recurrence and systemic spread. Thyroid involvement is infrequently reported, and generally seen in the context of Hashimoto thyroiditis. Additionally, amyloid deposition is an exceptional complication of extranodal MZL. We report an unusual case of a 65-year-old woman with a rapidly growing symptomatic nodule of the right thyroid lobe and a previous inconclusive FNA showing benign lymphoid tissue. She was taken to the OR for diagnostic right thyroid lobectomy with neck level 6 dissection. Methods/Case Report The specimen was sent for histologic evaluation, and showed thyroid with marked amyloid deposition, highlighted by Congo Red special stain, distorting normal architecture. Additionally, significant small atypical lymphoid infiltrate was present. Neoplastic lymphocytes were positive for CD20, PAX5 and CD43. Kappa light chain restriction by kappa ISH stain indicated monoclonality. Conversely, CD138, CD10, BCL6 and CyclinD were negative. Ki-67 demonstrated a 15-20% proliferation index. The germinal centers of the secondary follicles demonstrated diminished CD10 and BCL-6 consistent with follicular colonization by neoplastic lymphocytes. One of the regional lymph nodes displayed involvement by lymphoma with amyloid deposition. Medullary carcinoma of the thyroid was ruled out on the basis of incompatible histologic and immunohistochemical profile. Results (if a Case Study enter NA) NA Conclusion While Medullary carcinoma and amyloid goiter are among the top differentials for extensive amyloid deposition in the thyroid gland, we illustrate the need for judicious review of clinical history and careful consideration of immunohistochemistry workup to establish the unusual diagnosis of extranodal MZL of the thyroid with extensive amyloid deposition.

Abstract 13860: Heart Failure With Preserved Ejection Fraction Induced by Amyloid Light Chain Amyloidosis: A Case Report

Introduction: Cardiac amyloidosis is a clinical disorder defined as an extracellular deposition of protein in the heart. Typically, signs and symptoms of heart failure develop in the advanced disease. High index of suspicion is required for the diagnosis of this disorder. Results: A 62 years old male came to the cardiology clinic due to lower extremity edema, fatigue, dyspnea on exertion. He had a 3 month history of progressive lower leg edema. On physical examination he had a positive S4; lung sounds were diminished and there was dullness to percussion over the lower two thirds; and bilateral lower extremity edema. Hospital admission was decided for management of decompensated heart failure. On admission, the surface ECG showed normal sinus rhythm with low QRS voltage in the limb leads, a RBBB and LPFB. Initial laboratory reported elevated NT-proBNP: 13,816 pg/mL. Chest x-ray demonstrated right interstitial infiltration and bilateral pleural effusion. The echocardiogram revealed severe concentric hypertrophy of the left ventricle, an apical sparing left ventricle longitudinal strain, and an ejection fraction of 68%. A cardiac MRI showed concentric biventricular hypertrophy associated with extensive diffuse late gadolinium enhancement, suggestive of cardiac amyloidosis. Screening resulted in a Perugini score of 1 on Tc-PYP scintigraphy, elevated Kappa and Lambda light chains on serum, and kidney biopsy positive for amyloidosis. Hematology-oncology consultation was done and a daratumumab-CyBorD protocol was started. Conclusions: This case elucidated a stepwise diagnostic approach of cardiac amyloidosis in a patient presenting with signs and symptoms of heart failure. Amyloid AL cardiac amyloidosis and its repercussions are severe because it not only compromises by being infiltrative but also by its toxic capacity. A high index of suspicion and a multidisciplinary approach is needed in order to improve quality of life and prolong survival.

ODP541 Burr Under the Saddle: A Case of Sellar Plasmacytoma

Abstract Background Sellar and parasellar plasmacytoma is a rare tumor that often poses a diagnostic challenge as it can often be misdiagnosed as a nonfunctioning pituitary adenoma. Here we present a case of sellar plasmacytoma and point to clues helping establish the diagnosis. Clinical case 60-year-old male presented with two weeks of worsening morning headache followed by eyelid drooping. A physical exam was notable for left-sided ptosis, mydriasis, poor pupillary light reflex, and otherwise normal eye movement in cardinal positions . Head CT revealed 33×32×30 mm sellar mass; thus endocrinology team was consulted for evaluation. Further workup revealed normal TSH, free T4, ACTH and morning cortisol, FSH, LH, and IGF-1. He had a minimal elevation of prolactin levels at 33 ng/ml after serial dilutions (reference range: 2-17.7 ng/ml). We noticed the patient had an elevated serum protein concentration of 10.6 g/dl (reference range: 6.4-8.3 g/dl) with an albumin level of 4 gm/dl with an elevated gamma gap at 6.6 g/dl, suggestive of elevated gamma globulins. Serum protein electrophoresis showed an M-spike of 3.8 g/dl, and urine was positive for Bence Jones protein. Brain MRI with and without contrast showed homogeneously enhancing mass mainly within the sphenoid sinus extending down to the clivus, isointense on T1, hyperintense on T2 with no diffusion restriction, encasing the left internal carotid artery with intact pituitary and optic chiasm. Transsphenoidal biopsy showed sheets of plasma cells. Immunohistochemistry showed lambda light chain only and negative for kappa light chain. Flow cytometry was positive for CD38, CD138, CD200, and CD56, confirming a diagnosis of plasmacytoma. Fluorescent in situ hybridization was positive for FGFR3-IGH t(4;14) translocation, indicating a higher risk. Otherwise, beta-2-microglobulin, lactate dehydrogenase, and albumin were within normal. Initial management included pulsed dexamethasone of 40 mg for four days, followed by a taper with skull base radiotherapy, a total of 3500 cGy. Ptosis and eye movement gradually improved. The patient was discharged to start bortezomib-based treatment by the oncology team. Clinical lesson: Based on large case series, sellar plasmacytoma often presents as headache (70%), visual changes (77%), and hyperprolactinemia (38%). Imaging shows clinal invasion (74%), cavernous sinus invasion (49%), encasement of the internal carotid artery (17%) [1]. The differential diagnosis for such findings on imaging includes nonfunctioning pituitary adenoma, chordoma, and meningioma. Special attention to gamma gap, urinary, and serum protein electrophoresis can urge to perform a biopsy to prove the diagnosis of plasmacytoma and avoid delay in therapy. Reference: 1. Lee J, Kulubya E, Pressman BD, et al. Sellar, and clival plasmacytomas: case series of 5 patients with systematic review of 65 published cases. Pituitary. 2017;20(3): 381-392. doi: 10.1007/s11102-017-0799-5 Presentation: No date and time listed

Diagnostic Accuracy of Vitreous Cytology in Patients with Vitreoretinal Lymphoma.

(1) Background: To determine the diagnostic value of vitreous cytology in patients with vitreoretinal lymphoma (VRL) and evaluate its diagnostic accuracy relative to that of other diagnostic tests. (2) Methods: In total, 38 eyes from 38 patients with VRL who underwent diagnostic vitrectomy and were followed up for at least 6 months were analyzed. The clinical manifestations and VRL diagnostic rates for all diagnostic tests were determined. (3) Results: The presence of vitreous cells/opacity was the most common ophthalmic finding (97.4%), followed by sub-retinal pigment epithelial infiltration (65.8%) and retinal hemorrhage (21.1%). The VRL diagnostic rates were 89.3% for interleukin (IL)-10 levels &gt; 50 pg/mL; 82.1% for IL-10/IL-6 ratios &gt; 1; 60.0% and 63.3% for immunoglobulin heavy chain and kappa light chain clonality assays, respectively; and 44.4% for vitreous cytology. The VRL diagnostic rate for vitreous cytology was significantly lower in the steroid pretreatment group than in the non-steroid pretreatment group (p = 0.007). (4) Conclusions: The VRL detection rate for vitreous cytology was lower than that for the other tests, especially in patients who received steroid pretreatment. These findings suggest that even if vitreous cytology findings are negative, other tests and characteristic fundus findings should be evaluated to confirm VRL diagnosis.

Clinic-pathological characteristics of rare tubulointerstitial diseases.

Tubulointerstitial diseases is one of the common causes of renal dysfunction. Some rare pathological types are easy to be misdiagnosed and missedly diagnosed because of their low prevalence and relatively insufficient understanding, which affects the treatment and prognosis of patients. This study aims to explore clinical manifestations and pathological characteristics of several rare tubulointerstitial diseases, and therefore to improve their diagnosis and treatment. A total of 9 363 patients diagnosed by renal biopsy in the Department of Nephrology, Second Xiangya Hospital, Central South University from November 2011 to September 2021 were selected. Six cases of light chain cast nephropathy (LCCN), 2 cases of light chain proximal tubulopathy (LCPT), 1 case of LCCN with LCPT, 4 cases of genetic tubulointerstitial disease, and 6 cases of non-genetic related tubulointerstitial lesion were screened out, and their clinical manifestations and renal biopsy pathological results were collected, compared, and analyzed. Patients with LCCN presented with mild to moderate anemia, microscopic hematuria, and mild to moderate proteinuria. Compared with patients with LCPT, proteinuria and anemia were more prominent in patients with LCCN. Five patients with LCCN and 2 patients with LCPT had elevated serum free kappa light chain. Five patients with LCCN presented clinically with acute kidney injury (AKI). Two patients with LCPT and 1 patient with LCCN and LCPT showed CKD combined with AKI, and 1 LCPT patient presented with typical Fanconi syndrome (FS). Five patients with LCCN, 2 patients with LCPT, and 1 patient with LCCN and LCPT were diagnosed with multiple myeloma. Five patients with LCCN had kappa light chain restriction in tubules on immunofluorescence and a "fractured" protein casts with pale periodic acid-Schiff (PAS) staining on light microscopy. Immunohistochemical staining of 2 LCPT patients showed strongly positive kappa light chain staining in the proximal tubular epithelial cells. And monoclonal light chain crystals in crystalline LCPT and abnormal lysosomes and different morphological inclusion bodies in noncrystalline LCPT were observed under the electron microscope. Six patients with LCCN were mainly treated by chemotherapy. Renal function was deteriorated in 1 patient, was stable in 4 patients, and was improved in 1 patient. Two patients with LCPT improved their renal function after chemotherapy. Four patients with genetic tubulointerstitial disease were clinically presented as CKD, mostly mild proteinuria, with or without microscopic hematuria, and also presented with hyperuricemia, urine glucose under normal blood glucose, anemia, polycystic kidneys. Only 1 case had a clear family history, and the diagnosis was mainly based on renal pathological characteristics and genetic testing. Compared with patients with non-genetic related tubulointerstitial lesion, patients with genetic tubulointerstitial disease had an earlier age of onset, higher blood uric acid, lower Hb and estiated glomemlar fitration (eGFR), and less edema and hypertension. Renal pathology of genetic tubulointerstitial disease presented tubular atrophy and interstitial fibrosis, abnormal tubular dilation, glomerular capsuledilation, and glomerular capillary loop shrinkage. Glomerular dysplasia and varying degrees of glomerular sclerosis were observed. Genetic tubulointerstitial disease patients were mainly treated with enteral dialysis, hypouricemic and hypoglycemic treatment. Two genetic tubulointerstitial disease patients had significantly deteriorated renal function, and 2 patients had stable renal function. Patients with AKI or FS, who present serum immunofixation electrophoresis and/or serum free kappa light chain abnormalities, should be alert to LCCN or LCPT. Renal biopsy is a critical detection for diagnosis of LCCN and LCPT. Chemotherapy and stem cell transplantation could delay progression of renal function in patients with LCCN and LCPT. If the non-atrophic area of the renal interstitium presents glomerular capsule dilatation, glomerular capillary loop shrinkage, and abnormal tubular dilatation under the light microscopy, genetic tubulointerstitial disease might be considered, which should be traced to family history and can be diagnosed by genetic testing.

IgD multiple myeloma: biology, diagnosis, and treatment

IgD multiple myeloma is uncommon. Patients generally present at a younger age and have shorter progression free and overall survivals (OSs). Its rarity has inhibited development of a specific risk stratification system or informed best treatment protocols. We present interphase fluorescence in situ hybridization results from a group of 29 cases. These showed evidence of a decreased male to female ratio, decreased OS in patients aged 70 and over, better outcomes in those with kappa light chain restriction, and CD56 positive patients had longer survivals than those lacking CD56. We discuss the biology of IgD multiple myeloma, the need for prospective studies, and challenges for improvements in diagnosis and treatment. We suggest an International Register to accelerate development of best practice guidelines for diagnosis, risk stratification, and treatment.

Role of immunoglobulin heavy and light chain gene rearrangement analysis in differentiating between benign and malignant bone marrow B-cell lymphoid aggregates

Lymphoid aggregates are found in a minority of bone marrow biopsy and aspirate specimens, and when present, the distinction between benign and malignant aggregates can represent a diagnostic challenge. Morphologic and immunophenotypic evaluation of the aggregates can aid in that distinction but in a few cases, detection of immunoglobulin heavy chain (IGH) and kappa light chain (IGK) gene rearrangements may be needed to rule in or out a malignant disease process. We studied the role of testing for IGH/IGK rearrangements in the distinction between benign and malignant B cell-predominant lymphoid aggregates. Only a few studies have addressed this issue and most lacked an adequate number of cases for establishing statistical significance. Our study retrospectively evaluated 120 bone marrow aspirate and biopsy specimens, 79 cases originally diagnosed with benign lymphoid aggregates [4,5], and 41 demonstrating a B-cell lymphoma with malignant aggregates. Immunohistochemical stains were performed on all cases in our study and flow cytometry results were available in the vast majority of cases (98%). All patients included in our study but 9 had at least 2 years of clinical follow-up information. Of the malignant lymphoma cases, IGH/IGK rearrangements were demonstrated by polymerase chain reactionin 60% of the cases. Moreover, clonal rearrangements were identified in 15% of the cases with benign aggregates. After at least 2 years of follow-up, only one case with a positive clonality study occurring in the setting of morphologically benign-appearing bone marrow lymphoid aggregates experienced a relapse of non-Hodgkin lymphoma. Molecular analysis of the IGH and IGK genes may have utility in confirming the presence of malignancy in bone marrow aspirates and biopsy specimens. False-negative results, however, are possible due to testing limitations and sampling issues. Moreover, patients with conditions such as autoimmune disorders or infectious diseases are shown to also develop clonal B-cell lymphoid aggregates. As a result, we recommend a thorough morphological examination, informative immunohistochemical staining, accurate flow cytometric analysis, and current IGH/IGK rearrangement testing when evaluating bone marrow specimens containing B cell-predominant lymphoid aggregates, with the knowledge that molecular clonality results should be carefully interpreted in the context of morphological and immunophenotypic findings to prevent misdiagnosis.

Accuracy of determination of free light chains (Kappa and Lambda) in plasma and serum by Swedish laboratories as monitored by external quality assessment

BackgroundFree light chain (FLC) measurements are important in diagnosing monoclonal gammopathies. As FLC are heterogeneous, different reagents and instruments for measuring FLC concentrations may give diverging results that affect assessment of patients with monoclonal gammopathies. Here we investigated agreement between different FLC methods using data from the Swedish external quality assurance (EQA) programme. MethodsThe two main FLC assays, N Latex FLC (Siemens) and Serum Freelite (The Binding Site), using four nephelometric or turbidimetric instrument platforms, were compared. Results from 27 EQA rounds distributed to 11–16 Swedish hospital laboratories during 2015–2020 were investigated. ResultsThe kappa (κ) FLC measurements deviated significantly over time, but when only nephelometry was used, deviation from the mean was lower (median ranges: −5% to 13 %). The CV was significantly higher for the Freelite assay (mean CV = 8.7) than for the N latex assay (mean CV = 5.7) (p < 0.0001). The coefficient of determination between all combinations of reagents and instrument platforms used was generally good (r2 = 0.76–0.87), and the correlation slope acceptable (0.81–1.2). For lambda (λ) FLC measurements, no concordance between combinations of instruments and reagents is apparent, deviating between −40 % to + 48 % from the mean. The CV was significantly higher for the combination with nephelometry and the Freelite assay (CV mean = 13.9 %) than nephelometry and the N latex assay (CV mean = 9.9 %) (p <0.001). The coefficient of determination varied between combinations of reagents and instrument platforms (r2 = 0.59–0.89) and the slope ranged between 0.48 and 1.5. Significant differences between the two reagents used were sometimes noted. ConclusionsImprecision in λFLC affects the κFLC/λFLC ratio. This may be important in clinical assessment of patients, especially differentiating between monoclonal and polyclonal gammopathies.

Diving in the big blue: Atypical Mott cells in rapidly fatal primary amyloidosis.

A 75-year-old man with no previous medical history presented at the emergency unit with asthenia, anorexia, weight loss, and progressively increasing dyspnea revealing acute heart failure (N-terminal pro b-type natriuretic peptide:16 667 ng/L; troponemia: 47 ng/L). Echocardiography showed a granular appearance of the myocardium and interventricular septal hypertrophy, suggestive of amyloidosis. Chemistry disclosed high levels of immunoglobulin kappa light chains at 1240 mg/L (lambda light chains 14 mg/L; κ/λ ratio 86) and a detectable but unquantifiable IgA κ peak. Myocardial biopsies pathology showed an endocardium thickened by fibrosis. Within the myocardium, the presence of interstitial fibrillary deposits stained by Congo red and birefringent in polarized light confirmed the diagnosis of cardiac amyloid light-chain (AL) amyloidosis. The search for other organ damage disclosed vegetative involvement with orthostatic hypotension, icteric cholestasis (bilirubin 51.8 mg/L), undocumented digestive involvement (diarrhea with evocative endoscopic lesions), and glomerulonephritis (urinary proteinuria 4 g/24 h with 3 g/24 h albumin and 1 g/24 h immunoglobulin light chains). A bone marrow aspirate displayed 17% of plasma cells. Interestingly, a particular pattern of huge atypical Mott cells was identified (Figure 1A,B). The latter had so many cytoplasmic inclusions that the cytoplasm, and the nucleus could not be discriminated. These atypical cells are however plasma cells with an endoplasmic reticulum loaded with immunoglobulins and should be integrated as such in cell counts. Classical plasma cells and typical Mott cells with a few Russel bodies were also present (Figure 1C) indicating a continuum in the shape of the diseased plasma cells. Treatment with cyclophosphamide–bortezomib–dexamethasone-daratumumab was initiated in emergency. Unfortunately, the patient died 3 days later, of multiple organ failure. No additional septic or toxic causes could be identified. Mott cells are activated plasma cells containing intracytoplasmic inclusions with a “bunch of grapes” appearance. These inclusions, called Russell bodies, are dilated cisternae of the endoplasmic reticulum containing condensed immunoglobulins. They were identified in 1901 by the surgeon F.W Mott in the brains of monkeys with trypanosomiasis. Mott cells have been described in various pathological conditions: reactive plasmacytosis, monoclonal gammopathies, high grade B-cell lymphomas, Wiskott-Aldrich syndrome, and von Recklinghausen neurofibromatosis. AL amyloidosis is caused by clonal expansion of plasma cells producing misfolded immunoglobulin light chains responsible for the deposition of insoluble fibrillar proteins in many organs. Those most frequently affected are the kidneys, heart, liver, and peripheral nervous system. AL amyloidosis is a rare disease, and its diagnosis is often delayed. Although associated with a poor prognosis, AL amyloidosis presents a heterogeneous clinical expression and evolution. To our knowledge, the presence of Mott cells has rarely been described in this pathology. KR, ME, and BLC wrote the manuscript. KR, ME, and BLC performed biological analyses. CS treated the patient. All authors critically reviewed the manuscript. The authors have no conflict of interest to disclose. The authors received no specific funding for this work. The research was conducted in accordance with the principles embodied in the Declaration of Helsinki and in accordance with local statutory requirements.

Pasteurella multocida Toxin Aggravates Ligatured-Induced Periodontal Bone Loss and Inflammation via NOD-Like Receptor Protein 3 Inflammasome

NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is reportedly involved in periodontal pathogenesis. Pasteurella multocida toxin (PMT) is the major virulence factor of Pasteurella multocida strains, which belongs to the nonoral gram-negative facultative rods (GNFR). The existence of GNFR and their toxin may aggravate periodontitis. Therefore, it is important to unclose the regulatory mechanisms of PMT in periodontitis. However, the involvement of NLRP3 inflammasome and PMT in periodontitis remain unclear. The results showed that NLRP3 expression was increased in periodontitis mice by immunohistochemical staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Nlrp3-/- mice showed less periodontal bone loss and lower abundances of Pasteurella multocida by 16S rRNA sequencing. PMT promoted NLRP3 expressions by activating nuclear factor kappa light chain enhancer of B cells (NF-κB) pathway and activated NLRP3 inflammasome. This effect was reversed by NLRP3 inhibitor MCC950. Furthermore, PMT aggravated periodontal bone loss and inflammation in WT mice, while MCC950 attenuated periodontal bone loss and inflammation. The Nlrp3-/- periodontitis models with PMT local injection showed less bone loss and inflammation compared with WT periodontitis mice after PMT treatment. Taken together, our results showed that PMT aggravates periodontal response to the ligature by promoting NLRP3 expression and activating NLRP3 inflammasome, suggesting that NLRP3 may be an effective target for the treatment of periodontitis caused by GNFR and MCC950 may be a potential drug against this disease.

Saliva Free Light Chains in Patients with Neuro-Sjögren.

Background: Sjögren’s syndrome (SS) is an autoimmune disease characterized by sicca symptoms and various extra-glandular manifestations. The diagnosis of SS requires sicca symptoms, anti-SSA(Ro)-antibody positivity, and/or pathological focus scores on a minor salivary gland biopsy. Previous studies have investigated different biomarkers in order to avoid invasive diagnostic procedures. It was found that kappa and lambda free light chains (KFLC and LFLC) in saliva are specific for SS. Methods: FLC concentrations in saliva and serum were determined in 130 patients—50 with SS and neurological involvement (Neuro-Sjögren) and 80 neurological controls. The EULAR SS disease activity index and patient reported index (ESSPRI) were determined in patients with SS. Results: Patients with SS revealed increased pain and decreased saliva production according to the ESSPRI and Saxon test, respectively, with increasing FLC concentrations in the saliva. No significant differences in serum and salivary protein concentrations were observed between patients with SS and controls. Conclusion: KFLC and LFLC concentrations in saliva are not suitable to distinguish patients with Neuro-Sjögren and neurological control subjects, thus a diagnostic biopsy is still required. The association of salivary KFLC and LFLC concentrations with saliva production and ESSPRI pain score suggests a complex relationship between dryness and pain in patients with SS.

A CASE OF TYPE 1 CRYOGLOBULINEMIA PRESENTING WITH DIFFUSE ALVEOLAR HEMORRHAGE

A CASE OF TYPE 1 CRYOGLOBULINEMIA PRESENTING WITH DIFFUSE ALVEOLAR HEMORRHAGE

S3143 Rare Case of Plasma Cell Infiltration-Induced Cirrhosis

Introduction: Multiple myeloma (MM) is a type of malignancy that arises from the unorganized replication of plasma cells within the bone marrow. The overproduction of these cells damages systems throughout the body leading to early asymptomatic symptoms that can rapidly progress to systemic signs of disease. Liver involvement with MM is less common and generally occurs through plasma cell infiltration, light chain/amyloid deposition, and biliary obstruction. Though it can occur, the probability of it presenting near the time of diagnosis of MM is very rare and associated with poorer outcomes. Case Description/Methods: A 59-year-old male presented with generalized weakness, shortness of breath and weight gain. Physical exam showed sign of ascites and hepatomegaly. PMH was significant for esophageal cancer and a recent diagnosis of MM. No history of acute or chronic liver disease or cirrhosis risk factors. CT scan showed cardiomegaly, bilateral pleural effusions, pulmonary edema, and cirrhosis with abdominal ascites. Labs was unremarkable other than normocytic anemia. An echocardiogram showed mild reduced ejection fraction (47%) with speckle tracking suggestive of cardiac amyloidosis. Abdominal ultrasound confirmed appearance of cirrhosis, ascites, and right pleural effusion. Cirrhosis work up revealed negative hepatitis panel, normal alpha-1 antitrypsin, and normal ceruloplasmin. EGD was performed and ruled out extracellular vesicles (EV) but noted type I isolated gastric varices. Liver biopsy confirmed cirrhosis and showed portal tract damage with severe extensive plasma cell infiltration. Additionally, fragments of hepatic parenchymal showed disrupted lobular architecture by septal fibrosis and nodular formation. Immunohistochemistry was positive for CD3, CD20, CD5, BCL2, CD79a, and CD138 as well as increased lambda light chain compared to free kappa light chain. Despite extensive treatment including repeated thoracentesis and paracentesis, patient eventually developed in to decompensating liver cirrhosis (Figure 1). Discussion: In previous studies, plasma cell infiltration of the liver was seen in about 40% of cases of MM, but rarely lead to cirrhosis. This case is unique in that the patient had liver failure caused by cirrhosis and that it was some of the presenting symptoms. Whether due to masked signs or aggressive onset, the outcome for this diagnosis is poor and more specific factors observing liver function could have provided a more favorable outcome.Figure 1.: Cirrhosis secondary to plasma cell infiltration. Table 1. - Total Protein 5.0 g/dL Serum Ascites Albumin Gradient (SAAG) 1.9 g/dL Left Hepatic Vein Pressure 25 Hg Left Portal Vein Pressure 29 Hg Portosystemic Pressure Gradient 5 Hg EUS Guided Shear Wave Elastography of Liver 27 kPa